
flass l?S5 7 

Book Mj- 

CopightN 

COPYRIGHT DEPOSIT. 



CLINICAL 
LABORATORY METHODS 



CLINICAL 
LABORATORY METHODS 

A MANUAL OF TECHNIQUE AND MORPHOLOGY 

DESIGNED FOR THE USE OF STUDENTS 

AND PRACTITIONERS OF MEDICINE 



BY 

ROGER SYLVESTER MOHRIS, A.B., M.D. 

ASSOCIATE PROFESSOR OF MEDICINE IN WASHINGTON UNIVERSITY, ST. LOUIS. FORMERLY ASSOCIATE IN 
MEDICINE, THE JOHNS HOPKINS UNIVERSITY; ASSISTANT RESIDENT PHYSICIAN, THE JOHNS 
HOPKINS HOSPITAL; INSTRUCTOR IN MEDICINE AND DEMONSTRATOR OF 
CLINICAL MEDICINE, THE UNIVERSITY OF MICHIGAN. 




D. APPLETON AND COMPANY 
NEW YORK AND LONDON 
1913 






A 



A-\ 



Copyright, 1913, by 
D. APPLETON AND COMPANY 



Printed in the United States of America 



©CIA343997 
1Ul 



TO 
WILLIAM SYDNEY THAYER 

AND 

GEORGE DOCK 

THIS VOLUME IS DEDICATED 

WITH THE AFFECTION AND GRATITUDE OF 

THE AUTHOR 



PBEFACE 



Coincident with the improvement in medical education in this 
country there has been a .widespread increase in the use of labora- 
tory methods as aids to diagnosis. Not only is this true in the 
case of the more recent graduates in medicine, but, what is more 
hopeful, the older practitioners — those whose college days pre- 
ceded the introduction of Clinical Pathology into the medical cur- 
ricula — are quite generally realizing the necessity of the laboratory 
in their daily work. Probably no one thing has done more to 
bring about this much desired result than the discovery by Wasser- 
mann and his co-workers of the well-known serum reaction for 
the diagnosis of syphilis; unconsciously, perhaps, but none the 
less effectively, attention has been focused upon laboratory diag- 
nostic methods. 

The present volume is not a text-book of Clinical Pathology; it 
is a manual of laboratory technique and morphology, dealing merely 
with methods and with morphological elements which are of diag- 
nostic importance. It attempts to give in detail the means of de- 
tecting the abnormal in urine, gastric contents, feces, blood, spu- 
tum, and puncture fluids. Unlike the text-books, the significance 
of the abnormal is not discussed. 

That there is need for such a work the author has long believed. 
There is much which it is absolutely essential that the student of 
medicine — graduate and undergradute — remember. He must 
know, for example, under what conditions albuminuria may occur, 
whether it be of nephritic, cardiac, toxic, physiologic, or whatever 
origin. He must be aware of the possible significance of a second- 
ary anemia, of an atypical reduction test in the urine, of Charcot- 
Leyden crystals in sputa, of a hydrochloric acid deficit in the 
gastric contents. But it is useless to try to burden the memory 
with the details of the various laboratory methods, by which such 
abnormalities are detected, and with the sources of error in the 
methods. 



PREFACE 

Xo attempt has been made to include within the present volume 
a multiplicity of methods; in fact, the aim of author has been to 
select one method or more of proved value. Nor have the more 
exact, time-consuming methods of physiological chemistry been 
drawn upon; in his daily work the average clinician has not the 
time, if he has the ability, to employ them. 

Free use has been made of the following works: Emerson's 
" Clinical Diagnosis." Wood's "Chemical and Microscopical Diag- 
nosis," Simon's "Clinical Diagnosis," Sahli's ■'Klinische Unter- 
suehiingsinethoden. " Hoppe-Seyler's "Handbueh der chemischen 
Analyse." Hammarsten's "'LHkrbueh der physiologischen Chemie." 
Xeubauer-Huppert 's "Analyse des Haras." Schmidt and Stras- 
burger's "Die Faezes des Mensehen." Braun's '"Thierische Parasi- 
ten des Menschen." Blanc-hard's -'Traite de Zoologie Medieale." 
Cabot's "Clinical Pathology of the Blood." Xaegeli's "Blutkrank- 
heiten und Blutdiagnostik. " and Turk's "Vorlesungen ueber klin- 
ische Haematologie." Other authors have been consulted less 
freely. To the more recent literature direct reference has been 
given in the form of footnotes ; in all instances the writer has en- 
deavored to give proper credit to authors. 

It is hoped that the volume will prove helpful to medical 
students who have completed a course in Clinical Pathology and to 
practitioners of medicine, or that it may serve as a supplement 
to a course of laboratory lectures. 

For the original illustrations in black and white the author 
is indebted to Dr. James S. Brotherhood. It is a pleasure, also. 
to acknowledge his indebtedness to his wife and to his mother for 
assistance in the preparation of the index and in other ways. 

Roger Sylvester Morris. 
St. Louis. 



CONTENTS 

CHAPTER I 
THE URINE 

PAGE 

Collection of the Urine ....... 1 

Preservation of the Urine 2 

Macroscopic Examination of the Urine .... 3 

Color of Urine 4 

Quantity of Urine . . . 4 

Reaction of Urine 4 

Quantitative determination of urinary acidity: Folin's 

method . 5 

Specific Gravity 6 

Urea . ... . . . . . . 7 

Hiifner's hypobromite method ...... 8 

Uric Acid 9 

Qualitative determination of uric acid .... 9 
Quantitative determination of uric acid: Method of 

Folin and Shaffer 10 

Ammonia 12 

Quantitative determination of ammonia: Folin's method 
— the vacuum distillation method — the formalin titra- 
tion method — Shaffer's modification of Schlosing's 

method 13-19 

Nitrogen 20 

Kjeldahl's method for determination of total nitrogen . 20 

Chlorids 23 

Qualitative test . 23 

Quantitative determination of chlorids: Harvey's modi- 
fication of Volhard's method 24 

ix 



x CONTENTS 

PAGE 

Sulphates 26 

Obermayer's test . .27 

Jaffe's test ■ 27 

Albumin 28 

Qualitative tests: Heat and acetic acid test — Heat and 
nitric acid test — Heller's test — Potassium ferrocyanid 
and acetic acid test ....... 28-34 

Quantitative determination of albumin : Tsuchiya 's mod- 
ification of the Esbach method — Removal of albumin 
from the urine ........ 35-36 

Bence- Jones' Body 36 

Albumose • 37 

Glucose 38 

Qualitative tests: Trommer's test — Fehling's test — Al- 
men-Nylander 's test — The fermentation test — Cippo- 
lina's modification of the phenylhydrazin test . .38-42 
Quantitative estimation of glucose: Benedict's first 
method — Benedict's second method — Polariscopic deter- 
mination — Robert's specific gravity method — Measure- 
ment of carbon dioxid gas formed during fermentation 44-51 

Levulose 53 

Seliwanoff's test, as modified by Borchardt ... 53 

The phenylhydrazin test 55 

Maltose . . 56 

Lactose 56 

Saccharose 57 

Pentose 57 

The phloroglucin test 57 

The orcin test ......... 58 

Bial's modification of the orcin test .... 58 

Glycuronic Acid 59 

B. Tollens' test 60 

Alkaptonuria 61 

Acetone . . ......... 62 

Qualitative tests: Gunning's test — Lieben's test — Legal's 

test — Lange's test 62-64 

Diacetic Acid 65 

Gerhardt's test .65 




CONTENTS xi 

PAGE 

Diacetic Acid (Continued) 

Arnold's test • . . . . . . . . .66 

ft Oxybutyric Acid 67 

Black's test 67 

Hart's test 68 

Urobilinogen 70 

Ehrlich's alclehyd test 70 

Urobilin 70 

Spectroscopic determination ...... 71 

Schlesinger 's test 71 

Jaffe's test 72 

Bile Pigments 73 

Qualitative tests: Foam test — Gmelin's test — Rosen- 
bach's modification of Gmelin's test — Huppert's test — 
Hammarsten's test ....... .73-75 

Hematoporphyrin . . . . . . . . .76 

Garrod's test ......... 76 

Hemoglobin 77 

Spectroscopic determination . . . . 78 

The guiac test ......... 81 

Heller's test 82 

Teichmann's hemin-crystal test ..... 82 

The Diazo Reaction 83 

Chyluria 84 

Lipuria . . . 86 

Ferments in the Urine ....... 86 

Wohlgemuth 's method for the determination of diastase 87 

Determination of lipase according to Hewlett . . 88 

The Urinary Sediments 89 

The unorganized sediments : The quadriurates of sodium 
and potassium — Uric acid — Calcium oxalate — Calcium 
sulphate— Monocalcium phosphate — Hippuric acid — 
Cholesterin — Xanthin — Hematoidin — Tyrosin — 
Leucin — Cystin — Tricalcium and trimagnesium phos- 
phates — Calcium carbonate — Ammoniomagnesium phos- 
phate — Ammonium biurate — Neutral magnesium phos- 
phate — Neutral calcium phosphate ..... 91-99 



Xll 



CONTENTS 



The Urinary Sediments (Continued) 

The organized sediments : Epithelial cells — Pus — Blood — 
Casts — Mucous threads — ' ' Clap threads ' ' — Gonococcus 
— Treponema pallidum — Bacillus tuberculosis 

Animal Parasites in Urinary Passages 
Trichomonas vaginalis 
Filaria bancrofti 



Dioctophyrae renale . 

Schistosoma hematobium 
Prostatic Fluid 
Functional Dl\gnosis of the Kidney 

The phthalein test of Rovntree and Geraghty 



100-115 
116 
116 
117 
117 
118 
119 
119 
120 



CHAPTER II 



THE GASTEIC JUICE 



Test Breakfasts 125 

Examination of the Fasting Stomach . . . .127 
Macroscopic Examination of the Gastric Contents . . 128 

Quantity 128 

Odor 128 

Mucus 128 

Color 129 

Food . 129 

Chemical Examination of the Gastric Contents . '. 131 

Reaction 131 

Hydrochloric acid: Qualitative tests for free hydrochlo- 
ric acid (von den Yelden's methyl violet test: Giinz- 
berg's test; Tropeolin test; Congo-paper test: Topfer's 
test; Sahli's desmoid test) — Quantitative determination 
of gastric acidity (Topfer's method for free hydrochloric 
acid; Other indicators; Titration of total acidity) — The 

hydrochloric acid deficit 131-138 

Lactic acid: Qualitative tests for lactic acid (Ueffel- 
mann's test; Strauss' test; Kelling's test) . . 141-112 
Butyric acid 112 



CONTENTS xiii 

Chemical Examination of the Gastric Contents (Continued) 

PAGE 

Acetic acid 143 

Pepsin: Qualitative test for pepsin — Quantitative meth- 
ods (Mette's method as modified by Nierenstein and 

Schiff) 143-144 

Eennin: Qualitative test for rennin .... 145 

Eennin zymogen ........ 146 

Pathological enzyme in the gastric contents . . . 146 

Mucus . 148 

Microscopic Examination of the Gastric Contents . . 149 



CHAPTER III 
THE FECES 

Macroscopic Examination of the Feces . . . . 152 

Amount .......... 153 

Form 153 

Color .153 

Mucus 154 

Gallstones 155 

Parasites 155 

Intestinal Test Diet 155 

Diet No. I 156 

Diet No. II 156 

Weight of Dried Feces 157 

Chemical Examination of the Feces 157 

Reaction .......... 157 

Pigments . ......... 157 

Urobilin (Hydrobilirubin) : Schmidt's test — Schlesin- 

ger's test — Spectroscopic determination .... 158 

Bilirubin: Schmidt's test — Gmelin's test . . 158-159 
Blood: Weber's test — The guiac test — Teichmann's 

hemin crystal test 159-161 

Trypsin: Method of Gross for the determination of 

trypsin .......... 162 



XIV 



CONTEXTS 



PAGE 

Chemical Examination of the Feces (Continued) 

Amylase: Wohlgemuth 's method for the determination 

of amylase, as modified by Hawk ..... 163 

Microscopic Examination of the Feces .... 166 
Food remnants ........ 167 

Bacteria 168 

Cells 169 

Crystals 170 

Intestinal parasites: Protozoa, Rhizopoda — Flagellata — 
Infusoria — Nematodes — Trematodes — Cestodes . 172-194 

Preservation of gross specimens of cestodes and other 
parasites : Permanent preparations of flat worms 
(Method of Mink and Ebling — Boggs' method — Creosote 

method) 199-203 

Accidental contaminations ...... 203 

CHAPTER IV 
THE SPUTUM 



Amount . 








. 








205 


Reaction 








. 








205 


Character 
















205 


Odor 
















205 


Consistence . 








. 








205 


Air Bubbles . 








. 








205 


Dittrich's Plugs 








. 








205 


Bronchial Casts 








. 








206 


Ccrschm ann's Spirals 






. 








206 


Layer Formation . 






. 








206 


Microscopic Examination 




. 








206 


Examination of fresh spu 


turn 


. 








206 


Yellow elastic tissue 




. 








208 


Curschmann 's spirals 




. 








208 


Alveolar epithelial cells 












209 


Dust cells 












210 


' ' Heart-failure 


sells' 


? 












210 



CONTENTS xv 

PAGE 

Microscopic Examination {Continued) 

Red blood corpuscles 210 

Pus cells 211 

Eosinophilic leukocytes , 211 

Lymphocytes ......... 211 

Charcot-Leyden crystals . . . . . . .211 

Microorganisms in sputa: Bacillus tuberculosis (Ziehl- 
Neelsen method; Antiformin method for the detection of 
tubercle bacilli) — Diplococcus pneumoniae (Gram's 
method of staining; Welch's capsule stain) — Bacillus in- 
fluenzae — Bacillus diphtherias (Neisser's staining method; 
Beall's method) — Actinomyces bovis — Streptothrix ep- 

pengeri — Blastomycetes 212-223 

Animal parasites in the sputum: Entamoeba histolytica 
— Entamoeba tetragena — Trichomonads — Paragonimus 
westermanii — Echinococcus cyst .... 224-225 



CHAPTER V 

THE BLOOD 

Obtaining Blood for Examination 226 

Blood Stickers 226 

Counting the Blood Corpuscles 227 

The hemocytometer ........ 227 

Procedure in counting the erythrocytes : Diluting fluids — 
Filling the pipette — Filling the counting chamber — The 
enumeration of the cells — Calculation of the result — 

Cleaning the apparatus 229-234 

Counting the leukocytes: Diluting fluid — Filling the pi- 
pette — Filling the counting chamber — The enumeration 
of the leukocytes — Calculation of the result — Biirker's 
modification of the Thoma counting chamber . 236-239 
Counting the eosinophilic leukocytes: Dunger's method 241 
Counting the blood platelets: Method of Wright and 
Kinnicutt 242 



XVI 



CONTENTS 



PAGE 

Hemoglobin Determinations 244 

Tallqvist's method . 244 

Sahli's hemometer 245 

The Fleischl-Miescher hemoglobinometer . . . 249 
Haldane's hemoglobinometer ...... 252 

Dare's hemoglobinometer 252 

Sulphhemoglobinemia 252 

Methemoglobinemia 252 

Color Index 253 

Volume Index . . 253 

Measuring the Diameter of Cells 255 

Viscosity of the Blood and Other Fluids . . . 256 

Method of Hess 256 

The Specific Gravity of the Blood 259 

The Coagulation Time of the Blood 260 

Method of Brodie and Russell, as modified by Boggs . 260 
Milian's method, as modified by Hinman and Sladen . 263 
The Resistance of the Red Blood Corpuscles . . . 264 
The Examination of Fresh and Stained Preparations of 

Blood 265 

The cleaning of cover glasses and slides .... 265 
Examination of the fresh blood : Sealing the fresh speci- 
men — The preparation of dry (permanent) blood smears 

— Fixation of blood smears 265-271 

Staining the blood: Vital staining of the blood — 
Vaughan's method; Method of Widal, Abrami, and 
Brule ; The ' ' dry ' ' method of vital staining ; The stain- 
ing of dried blood films — Methylene blue ; Eosin ; Hema- 
toxylin; Carbol-thionin. Staining mixtures of two or 
more stains — Ehrlich 's triacid stain ; The Romanowsky 
stains (Wilson's stain, Leishman's stain, Giemsa's 
stain) ; Jenner's stain; Methyl green-pyronin mixture 
of Pappenheim; The iodin reaction of the leukocytes 

273-295 
Differential counting of the leukocytes: Normal leuko- 
cytes — Pathological leukocytes .... 296-299 



CONTENTS xvii 

PAGE 

The Examination of Fresh and Stained Preparations op 
Blood (Continued) 
The normal and pathological red blood corpuscles: 
Erythrocytes — Erythroblasts — Abnormalities in the 
staining of the red corpuscles .... 301-302 
Demonstration of protozoa in the blood: Malarial para- 
sites . 305 

Examination of the blood for animal parasites: Filaria 
bancrofti — Trichinella spiralis ...» 308-310 



CHAPTER VI 

PUNCTURE FLUIDS 

Specific Gravity 312 

Albumin Content 312 

Incoagulable Nitrogen 313 

A Protein Precipitable in the Cold by Dilute Acetic 

Acid 315 

Cytology 315 

Cerebrospinal Fluid 318 

Cells of the cerebrospinal fluid 318 

Method of counting the cells 318 

Bacteriology of the cerebrospinal fluid .... 319 

Globulin content: Method of Noguchi — Method of Ross 
and Jones ........ 319-321 

The Wassermann reaction in cerebrospinal fluid . . 321 

Index 323 



LIST OF ILLUSTRATIONS 



FIG. PAGE 

1. — Apparatus for the quantitative determination of am- 
monia according to Folin 14 

2. — Apparatus for the determination of ammonia according 

to Shaffer . . . 16 

3. — Digesting rack for the Kjeldahl nitrogen determination 21 
4. — Distilling apparatus for the Kjeldahl nitrogen deter- 
mination 22 

5. — Lohnstein's areometer ....... 51 

6. — Lohnstein's fermentation saccharometer for undiluted 

urine 52 

7. — Absorption spectra 79 

8. — The Sydenham sedimenting glass 89 

9. — Treponema pallidum; Spirochaeta refringens . . 112 

10. — Embryo of Filaria bancrofti ...... 117 

11. — Ova of Dioctophyme renale ...... 118 

12. — The Autenrieth-Konigsberger colorimeter as modified by 
Rowntree and Geraghty for the determination of 

phenolsulphonephthalein ...... 123 

13. — Parasitic amebaa 173 

14. — Trichomonas intestinalis 177 

15. — Lamblia intestinalis 178 

16. — Balantidium coli 179 

17. — Ovum of Necator americanus . . . . . .. 181 

18. — Necator americanus . . . . . . . 185 

19. — The rhabditiform embryo of Strongyloides stercoralis . 186 

20. — Ovum of Strongyloides stercoralis 187 

21. — The rhabditiform embryo of Strongyloides stercoralis 

and the embryo of the hookworm .... 188 

22. — Ovum of Oxyuris vermicularis 188 

23. — Ovum of Trichuris trichiura 189 



xix 



xx LIST OF ILLUSTRATIONS 

FIG. PAGE 

24. — Ovum of Ascaris lumbricoides : tlie same under high 

focus, showing the albuminous coating . . . 190 

25. — Unfertilized ovum of Ascaris lumbricoides . . . 191 

26. — Ovum of Schistosoma haematobium .... 193 

27. — Ovum of Schistosoma japonicnm 193 

28. — Gravid proglottis of Tenia saginata : ovum of Tenia 

saginata ; gravid proglottis of Tenia solium . . 195 

29. — Ovum of Dibothriocephalus latus 196 

30. — Gravid proglottis of Dibothriocephalus latus . . . 197 

31. — Ovum of Hynienolepis nana ...... 197 

32. — Ovum of Hynienolepis diminuta ..... 198 

33. — Dipylidium caninum. showing an egg capsule and a 

free ovum 199 

34. — Tyroglyphus siro. the cheese-mite and ovum . . . 2'~>2 
35. — Actinomyces hominis. showing club-shaped extremities 

to the rays 222 

36. — Blastomycetes in sputum 223 

37. — Ovum of Paragonimus westermanii from the sputum . 224 

38. — Sediment from echinococcus cyst 225 

39. — The Thoma-Zeiss hemocytometer ..... 227 

■40. — The Xeubauer ruling of the hemocytometer . . . 22 S 

41. — Biirker's hemocytometer 239 

42.— The Sahli hemometer 245 

43. — The Fleischl-Miescher hemoglobinometer . . .250 
44. — The viscosimeter of Hess . . . . . .257 

45. — Boggs' modification of the coagulometer of Brodie and 

Russell 261 

46. — Diagram to illustrate the movement of the cells during 

coagulation 262 



CLINICAL LABORATORY 
METHODS 

CHAPTER I 

THE URINE 

Collection of the Urine.— For chemical examination, as 
a general rale, the total amount of urine for the twenty- 
four hours should be preserved. The reason for saving all 
the urine is that different voidings may vary greatly in 
their chemical composition. In the morning, for example, 
an albuminuric may excrete urine which is normal chemi- 
cally, whereas specimens obtained after the patient has 
had more or less exercise may contain albumin. Thus, it 
becomes necessary that a mixture of all the urine passed 
during the twenty-four hours be obtained in order to avoid 
the possibility of error, for the examination of the early 
morning specimen alone, in the case just cited, would be 
entirely misleading. Special circumstances arise at times, 
nevertheless, which make it desirable to break the rule and 
to secure one or more voidings at special hours of the day. 
For the purpose of quantitative chemical analysis, it is, of 
course, absolutely essential to have the total amount of 
urine for twenty-four hours collected. 

For microscopic examination a perfectly fresh specimen 
should always be insisted upon. The organized elements 
of a urinary sediment rapidly deteriorate, especially with 
high temperatures, so that within a few hours after the 
urine has been passed they may be unrecognizable, or com- 
pletely disintegrated. When it is not possible to make an 

1 



2 PRESERVATION OF THE URINE 

immediate examination a preservative (thymol, toluol) 
should be added to the specimen, which is kept in an ice- 
chest as a further safeguard. 

Preservation of the Urine.— To all twelve or twenty- 
four-hour specimens of urine a preservative should be 
added to prevent decomposition through bacterial growth. 
The urine should also be kept on ice when possible. The 
receptacle for the urine must be perfectly cleaned and 
tightly stoppered. 

(1) Toluol (toluene) is, on the whole, one of the most 
satisfactory preservatives. It apparently interferes with 
none of the urinary tests. Bacterial growth is successfully 
inhibited by means of it. The one disadvantage in its use 
results from the fact that toluol floats on the urine; it is 
necessary to pipette or siphon the urine to obtain it with- 
out admixture of the preservative. The objection is a 
minor one. Diacetic acid may be preserved for weeks, 
whereas it disappears in a short time when other preserva- 
tives are used. In acidosis, therefore, toluol should be used 
as the preservative. Organized sediments are often beau- 
tifully preserved, but it must be remembered that casts or 
cells, becoming attached to droplets of toluol, rise to the 
surface and may be missed, when only a few are present; 
spontaneous sedimentation cannot be relied on if toluol 
is employed. The pipette used for withdrawing the sedi- 
ment must be wiped to remove the toluol before placing the 
drop on a slide for examination. 

(2) Chloroform is the most generally used preservative 
for chemical work. It is a fairly strong reducing agent, 
and urine preserved with it must be boiled to drive it off 
before any of the reduction tests for sugar are performed. 
If the sediment is to be examined, care should be exercised 
to avoid drawing up chloroform with it. 



THE URINE 3 

(3) Thymol is very satisfactory, and with it the formed 
elements of the nrine are often very well preserved. As 
Weinberger * has shown, many urines to which thymol has 
been added give a positive Heller's test, though albumin 
be absent, a source of error which must be kept in mind. A 
positive test for bile may also be obtained after thymol 
preservation (Emerson). 

Gum camphor and formaldehyde are used occasionally 
as preservatives. Formaldehyde, like chloroform, is a re- 
ducing agent. When available, toluol, chloroform, or thy- 
mol is to be preferred. 

Macroscopic Examination of the Urine.— As a general 
rule, normal, freshly voided urine is perfectly clear; the 
same is true of the majority of pathological urines. Occa- 
sionally, if the reaction of the urine be alkaline when 
voided, a turbidity may result from the precipitation of the 
phosphates and carbonates in the bladder, in the absence 
of a cystitis. Ordinarily, however, fresh urine, when cloudy 
or turbid, contains pathological ingredients, such as blood, 
pus, bacteria in large number, phosphates, etc. Normal 
and pathological urines will become turbid and produce 
a macroscopic deposit, more or less abundant, if allowed 
to stand for some hours. Concentrated urine often fur- 
nishes an abundant precipitate of urates on cooling; the 
urates may be redissolved by warming the specimen. More 
frequently bacterial decomposition is the cause of the tur- 
bidity. 

The nubecula is a translucent cloud, composed chiefly 
of mucin (mucous threads) enmeshing epithelial or other 
cells, which forms in the urine a short time after it is 
passed. 

1 Weinberger, W. ' ' Thymol as a source of error in Heller 's test for 
urinary protein." Jour. A. M. A., 1909, LII, 1310. 



4 REACTION OF URINE 

The color of the urine is usually dependent on the quan- 
tity of water excreted in the twenty-four hours; the smaller 
the amount of urine the deeper the color, and vice versa. 
Normal urinary pigments in increased concentration or 
pathological pigments may lead to abnormal coloration of 
the urine (see urobilin, bilirubin, hemoglobin, hematopor- 
phyrin, etc.). Following the administration of certain 
drugs, the color of the urine may be altered, the most strik- 
ing change being the green color produced by methylene 
blue. 

Quantity of Urine. — The normal average amount of 
urine for the twenty-four hours in this country is about 
900 to 1,200 c. c. The limits of the normal are said to be 
800 to 3,000 c. c. (Emerson). In health the quantity de- 
pends chiefly upon two factors, the amount of water con- 
sumed and the amount lost by perspiration. In disease the 
quantity of urine passed in twenty-four hours may be nor- 
mal, increased, decreased, or nil. 

In certain diseases the urine is saved to advantage in 
twelve-hour periods, 7 A. M. to 7 P. M., and 7 P. M. to 7 
A. M. In health the ratio of the quantity of the day urine 
is to that of the night urine as 67 :33, though it may be as 
50:50, considering the total amount for twenty-four hours 
as 100. In disease the quantity voided during the night 
may exceed that for the day, as Edmunds 1 and others have 
shown. 

REACTION OF URINE 

The reaction of the urine is usually slightly acid, owing 
to the presence of an excess of dihydrogen (diacid) phos- 
phates. An amphoteric reaction (red litmus turned blue 
and blue turned red) may be encountered, due to the fact 

1 Edmunds, C. W. ' ' Observations on the quantity of day and night 
urine." N. Y. Med. Jour., 1904, LXXIX, 245. 



THE URINE 5 

that mono sodium phosphate, an acid salt, may exist in the 
urine in conjunction with disodium phosphate, which is 
alkaline. An alkaline reaction is produced largely by an 
excess of alkaline phosphates and carbonates. That the 
salts are not the only factor in rendering a urine acid has 
been shown by Folin, who finds that at times nearly half of 
the acidity may be due to organic acids. 

Litmus paper is used in testing the reaction of the urine. 
Unpreserved specimens, which have been allowed to stand 
for some time before testing, are often alkaline from am- 
moniacal fermentation produced by bacteria. The alkalim 
ity in this case is differentiated from that due to fixed alkali 
by the odor, by the fact that on boiling the specimen the 
steam will turn blue a piece of moistened red litmus held 
in the neck of the test tube, or will cause a white frost of 
ammonium chlorid to appear on a glass rod, which has been 
dipped in hydrochloric acid. In disease the urine may be 
ammoniacal before it is voided. 

Quantitative Detekmination of Urinaky Acidity 

For quantitative determination of the acidity the twen- 
ty-four-hour specimen is used. It is necessary to prevent 
decomposition by the addition of a preservative. 

Folin's Method. 1 

Eeagents : 

^-sodium hydrate. 2 

0.5 per cent, phenolphthalein in 50 per cent, al- 
cohol. 
Potassium oxalate, neutral. 

1 Folin, O. "The acidity of the urine." Amer. Jour. Physiol., 1903, 
IX, 265. 

2 A normal solution of acid or alkali should be purchased from a reliable 
firm. With this as a standard, the physician may easily prepare most of the 
remaining normal solutions required in routine work. 



6 SPECIFIC GRAVITY 

Method. — "With a pipette transfer 25 c. c. of urine into 
a small Erlenmeyer flask (capacity 200 c. c). Add one or, 
at most, two drops of phenophthalein and 15 to 20 gms. 
powdered potassium oxalate. Shake about one minute and 
titrate at once with tenth normal hydrate until a faint, yet 
distinct, pink coloration is produced throughout the con- 
tents of the flask. Shaking should be continued during the 
titration, so as to keep the solution as strong as possible in 
oxalate." The number of cubic centimeters of sodium hy- 
drate used multiplied by 4 gives the acidity per cent, in 
terms of tenth normal alkali. 

The inaccuracy of direct titration of the urine with 
sodium hydrate, as proposed by Naegeli, is pointed out by 
Folin. The two chief sources of error are ammonium salts 
and the occurrence of calcium in the presence of acid phos- 
phates. By first treating the urine with potassium oxalate 
each of these sources of error is practically eliminated. 

Normal values with this method are 25 to 30 acidity per 
cent. (Wood). 

SPECIFIC GRAVITY 

As a rule, the determination of the specific gravity of 
the urine is of real value only in the twenty-four-hour speci- 
men. It is usually determined by means of an urinometer. 
The short, small instruments designed for the purpose of 
taking the specific gravity of small quantities of urine are 
usually very inaccurate. 

In using the urinometer the urine is carefully poured 
into a glass cylinder, so that no foam is produced. Should 
foam collect despite the precautions, even though there be 
only a few bubbles, they should be removed with filter 
paper. The cylinder must be sufficiently wide to permit the 
urinometer to float freely without coming in contact with 



THE URINE 7 

its wall. The reading- is made with the eye on a level- with 
the bottom of the meniscus (the concave upper surface of 
the fluid). The instruments are standardized for use at a 
temperature of 15° C. ordinarily. For each 3° C. above this 
temperature the specific gravity is depressed one point in 
the third decimal place. As an example, if the specific 
gravity of a urine were found to be 1.015 at 24° C, the cor- 
rected reading would be 1.015+0.003=1.018. 

In case the specimen of urine furnished for examination 
be small, the urine which remains after the necessary tests 
have been performed may be diluted with water and the spe- 
cific gravity of the diluted specimen determined. The last 
two figures of the specific gravity found are multiplied by 
the dilution ; the result approximates the specific gravity of 
the undiluted urine. 

Normally the specific gravity of the twenty-four-hour 
specimen varies between about 1.010 and 1.025; absolute 
limits for the normal cannot be assigned, for so many fac- 
tors enter into the determination of the specific gravity 
that in individual instances the figures given may be passed 
in either direction, without necessarily signifying disease. 
It must be remembered that readings made with the urino- 
meter are not absolutely correct, but are sufficiently accu- 
rate for clinical purposes. Where greater accuracy is re- 
quired a pycnometer should be employed. 

An approximate idea of the amount of solids dissolved 
in the urine may be obtained by multiplying the last two 
figures of the specific gravity by 2.33 (Haser's coefficient), 
the result being the amount of solids in grams. 

UREA 

The normal amount of urea excreted daily in the urine 
varies within rather wide limits. Values between 20 and 



8 UREA 

40 gms. are usually found. In clinical work urea deter- 
minations have been j3ractically abandoned, except in the 
diagnosis of unilateral renal disease. 

Hiifner's Hypobromite Method.— The most convenient 
apparatus for applying this test is Heinz 's modification of 
the Doremus tube. It consists of a J-shaped tube mounted 
on a stand. A bulb is blown in the extreme end of the 
"tail" of the J- tube and a second tube of 2 c. c. capacity, 
graduated in _i_ c. c, is blown into the upright arm of 
the J-shaped tube. The connection between the two tubes 
may be cut by means of a glass cock in the 2 c. c. tube. The 
upper end of the J-tube is sealed. 

The reagent, Eice's bromin solution, is prepared as fol- 
lows: 



Sol. 1. Sodium hydrate 40.0 gm. 

Distilled water 100.0 c. c. 



Sol. 2. Bromin 10.0 c. c. 

Potassium bromid 10.0 gm. 

Distilled water 80.0 c. c. 

The two solutions are kept in separate bottles, and at 
the time of performing the test are mixed in equal volumes. 

Method. — Fill the small tube with the urine. The stop- 
cock is then opened until the urine reaches the zero mark. 
The excess of urine, which has run into the large J-tube, is 
removed from the latter by washing it with water, the up- 
per end of the tube containing the urine being appropri- 
ately sealed to prevent its escape. The J-tube is now filled 
with the mixed solutions, sufficient of the latter being em- 
ployed to completely fill the upright (all air must be dis- 
placed). The stop-cock is opened and the urine is slowly 
run into the mixed solutions. As the two fluids come in 



THE URINE 9 

contact, the hypobromite liberates nitrogen gas, which col- 
lects at the upper end of the large tube. The volume of 
gas liberated by 1 c. c. of urine is read on the scale marked 
on the upright arm of the J-tube, and gives the urea in 
grams in 1 c. c. of urine. 

The method is very inaccurate as a means for deter- 
mination of urea ; the results obtained approach more near- 
ly the total nitrogen of the urine. For this reason the 
method is inapplicable, where exact values for urea are re- 
quired, as, for example, in metabolism experiments. In the 
diagnosis of surgical affections of the kidney, where the 
urine from each kidney is examined separately, marked dif- 
ferences in the two kidneys are shown with sufficient ac- 
curacy, and it is in this connection that the method is used 
most at the present time. 

URIC ACID 

The normal quantity of uric acid in the urine in twenty- 
four hours lies between 0.1 and 1.25 gm., with a patient on 
a mixed diet. The endogenous uric acid of the urine varies 
between 0.1 and 0.4 gm. 

Qualitative Deteemination of Ukic Acid 

Qualitative determination of uric acid is made by the 
murexid test. A small drop of the urinary sediment or 
other material to be tested is dissolved in two or three 
drops of nitric acid in a porcelain evaporating dish. The 
material is evaporated to dryness, preferably on a water 
bath, care being exercised to avoid burning the prepara- 
tion. The stain which remains on the dish has a reddish 
color. (A yellow stain may indicate that an insufficient 
quantity of nitric acid was used.) The addition of ammo- 



10 URIC ACID 

nium hydrate or, better still, exposing the stain to ammonia 
fnmes changes the color to a purplish red, which fades on 
heating. The reaction is given by uric acid and by its salts. 
(For further qualitative tests, see urinary sediments.) 

Quantitative Determination of Uric Acid 

Method of Folin and Shaffer. 1 
Eeagents : 

Sol. 1. Ammonium sulphate 500.0 gm. 

Uranium acetate 5.0 gm. 

Distilled water to 650.0 c. c. 

Dissolve and then add : 

Acetic acid, 10 per cent 60.0 c. c. 

Distilled water to 1,000.0 c. c. 

Sol. 2. Ammonium sulphate 100.0 gm. 

Distilled water to 1,000.0 c. c. 

Sol. 3. ^jr potassium permanganate. 

To prepare the twentieth normal permanganate solu- 
tion, dissolve 1.7 gm. of potassium permanganate in one 
liter of distilled water. The solution is boiled or auto- 
claved to render it more permanent. After it has cooled 
to room temperature, it is titrated against tenth normal 
oxalic acid solution (6.3 gm. pure crystals to one liter of 
distilled water). With a pipette, 10 c. c. of _n. oxalic acid 
are placed in a small Erlenmeyer flask or beaker, diluted 
with about 100 c. c. of distilled water, and 15 c. c. of concen- 
trated sulphuric acid added. The temperature of the mix- 
ture is raised, by the addition of the sulphuric acid, to about 

1 Folin, O., and Shaffer, P. A. "Ueber die quantitative Bestimmung der 
Harnsaure im Harn. " Ztschr. f. physiol. Chem., 1901, XXXII, 552. 



THE URINE 11 

60° C. While still hot the permanganate solution is added 
to it from a burette, under constant stirring, until a uni- 
form red color appears, which persists throughout the fluid 
for a few seconds. 1 This is the end reaction. The quan- 
tity of permanganate solution used is read off on the bu- 
rette. Since the permanganate solution has been made too 
strong, less than 20 c. c. of it should have been required to 
produce the end reaction. The permanganate solution 
which remains is accurately measured (with volumetric 
flasks and pipettes, not with cylinders), and is diluted with 
distilled water, so that exactly 20 c. c. will give the end re- 
action with 10 c. c. of tenth normal oxalic acid. When kept 
in a dark place, tightly stoppered, the potassium perman- 
ganate solution is fairly permanent for several months. 
The titer should, however, be determined from time to time 
with the oxalic acid solution. 

Example. — If 18.9 c. c. of permanganate solution gives 
the end reaction with 10 c. c. of n oxalic acid, and the 
remainder of the potassium permanganate solution amounts 
to 960 c. c, the necessary dilution to make the perman- 
ganate solution twentieth normal is determined by the fol- 
lowing equation : 18.9 :20 : :960 :x. x=l,015.8. Therefore, 
the amount of water necessary to add would be 1,015.8—960 
or 55.8 c. c. 

Method. — To 300 c. c. of urine in an Erlenmeyer flask 
or beaker of 500 c. c. capacity or larger, add 75 c. c. of the 
uranium acetate reagent (sol. 1) to precipitate phosphates 
and other substances, which might interfere with the accu- 
racy of the method. Both urine and reagent must be accu- 
rately measured with volumetric pipettes. Stir the mixture 

1 Early in the titration, after the addition of the first few drops of 
permanganate, a red color, which may persist for fifteen seconds or so, may 
be noted, but it quickly disappears on adding more permanganate. 



12 AMMONIA 

well, and allow it to stand five minutes. Then filter through 
a double folded filter. Measure with a pipette 125 c. c. of 
the filtrate | this represents 100 e. e. of the urine originally 
used) into each of two beakers and add 5 c. c. of ammonium 
hydrate to each to convert the uric acid into ammonium 
urate. Mix well, and set aside for twenty-four hours ; by 
the end of this time the precipitate will have settled to the 
bottom of the beaker. The clear, supernatant fluid is de- 
canted, the precipitate collected on a filter ( Schleicher and 
SchiiU's Xo. 597) and washed with 10 per cent, ammonium 
sulphate, till the filtrate is almost chlorin free. (In testing 
for chlorids add a little nitric acid and then a few drops of 
dilute silver nitrate solution (10 to 15 per cent, solution); 
a white precipitate or cloud is formed if chlorids are pres- 
ent. ) The filter paper is now pierced with a glass rod, and 
the precipitate washed into a beaker with about 100 c. c. of 
distilled water. Add 15 c. c. of concentrated sulphuric acid 
and titrate the mixture immediately, stirring constantly, 
until a pink color appears throughout the fluid and persists 
for a few seconds. 

Each cubic centimeter of twentieth normal permanga- 
nate is equivalent to 3.75 mg. of uric acid. This, multiplied 
by the number of c. c. of permanganate used, gives the 
uric acid in 100 c. c. of urine, from which the total amount 
for twenty-four hours is readily calculated. Since ammo- 
nium urate is slightly soluble in water and somewhat more 
so in urine, a correction of 3 mg. should be added to the 
final result for each 100 c. c.of urine. 

AMMONIA 

Normally the urine contains 0.6 to 0.8 gm. of ammonia 
dailv for the average adult on a mixed diet. The limits of 



THE URINE 13 

the normal are about 0.3 to 1.2 gm. In health the ammonia 
nitrogen usually amounts to 4 to 5 per cent, of the total 
nitrogen, when a mixed diet is taken. 

Quantitative Determination of Ammonia 

(1) Folin's Method. 1 

Eeagents : 

Sodium chlorid. 

Anhydrous sodium carbonate. 

Petroleum or toluene. 

JL sulphuric acid ; _n_ sodium hydrate. 

One per cent, aqueous solution of alizarin red. 

The apparatus required includes areometer cylinders 
(45 cm. deep and 5 cm. in diameter), a suction pump, cal- 
cium chlorid tube, doubly perforated stoppers to fit the cyl- 
inders, and tubing for connections. Folin's tube to secure 
thorough mixing of air and acid is a convenience, though 
not a necessity. The apparatus is connected as shown in 
the illustration (Fig. 1), the calcium chlorid tube filled with 
cotton being placed between the cylinders to prevent the al- 
kaline urine being drawn over into the acid. 

Method. — With a pipette, 25 c. c. of tenth normal acid 
are placed in cylinder B and diluted with distilled water 
sufficiently to cover the end of the mixing tube. Into cylin- 
der A, 25 c. c. of the twenty-four-hour specimen of urine are 
measured with a pipette. To the urine are added 8 to 10 
gm. of sodium chlorid, 5 to 10 c. c. of toluol or petroleum 
to. prevent foaming (with blood or other fluid rich in pro- 
tein add some methyl alcohol also), and, finally, about 1 gm. 

1 Folin, O. ' ' Eine neue Methode zur Bestimmung des Ammoniaks im 
Harne und anderen thierischen Fliissigkeiten. " Ztsclir. f. physiol. Chem., 1902- 
03, XXXVII, 161. 



14 



AMMONIA 



of anhydrous sodium carbonate. After the addition of the 
soda the cylinder is immediately stoppered and the air cur- 
rent started. Before entering the urine the air current 
may be passed through a wash bottle containing sulphuric 
acid to remove ammonia in the air, though usually this pre- 
caution is unnecessary. 



u 



n 



A 



B 




^ _^ 



Fig. 1. — Apparatus for the Quantitative Determination of Ammonia According 
to Folin. Cylinder A for urine, cylinder B for acid. 



The addition of the soda to the urine liberates the 
weaker base, ammonia, which is carried over by the air 
stream into the tenth normal sulphuric acid, by which it 
is neutralized. When Folin 's mixing tube is used, not a 
trace of the ammonia escapes neutralization by the acid, 
even though there remains an excess of only 5 c. c. of tenth 
normal acid. If ordinary glass tubing be employed to pass 
the air through the acid, a second cylinder, containing 10 
c. c. of tenth normal acid, should be interposed between the 



THE URINE 15 

acid cylinder and the pump to catch the ammonia which es- 
capes neutralization. 

The air pump should be capable of carrying 600 to 700 
liters of air per hour through the apparatus. With a pump 
of this capacity working at room temperature (20 to 
25° C.) all of the ammonia is carried into the acid in an 
hour or an hour and a half. 1 

When the process is completed the acid is poured into 
an Erlenmeyer flask or beaker and the cylinder rinsed with 
distilled water, which is added to the acid. The acid is now 
titrated with tenth normal sodium hydrate, using two drops 
of alizarin red to 200 to 300 c. c. of fluid. The end reaction 
is the appearance of a red color throughout the fluid; do 
not continue the titration to the appearance of a. violet 
color. The difference between the number of cubic centi- 
meters of acid originally taken and that of the alkali used 
is the number of cubic centimeters of acid neutralized by 
ammonia. Since one cubic centimeter of tenth normal acid 
is equivalent to 0.0017 gm. of ammonia, this, multiplied by 
the number of c. c. of acid neutralized, gives the quantity of 
ammonia in 25 c. c. of urine. The quantity in the twenty- 
four-hour specimen is calculated from this. 

Determinations may be made in duplicate or triplicate 
by connecting two or more sets of apparatus in series. 

(2) The Vacuum Distillation Method. -Shaffer 2 has 
modified the vacuum distillation method. As described by 
him, it is carried out in the following manner : To 50 c. c. 
of urine in flask A (Fig. 2) add an excess (15 or 20 gm.) 

1 The efficiency of the pump and its ' ' working time ' ' are readily tested 
by taking a specimen of urine and titrating the acid at the end of an hour; 
add more tenth normal acid and titrate at fifteen-minute intervals, until acid 
is no longer neutralized. 

2 Shaffer, P. "On the quantitative determination of ammonia in urine." 
Amer. Jour. Physiol, 1903, VIII, 330. 



16 



AMMONIA 



lCITOj 



of sodium chlorid, and about 50 c. c. methyl alcohol. In 
bottle B place 25 or 50 c. c. ^ acid and in B' 10 c. c. -^ 
acid, diluted in each case with a small amount of water. 
If too much water is added there will be danger of loss of 
acid by jumping over during the violent commotion which 
is set up in the acid by the rapid passage of the steam. If 
such a loss should occur the acid can always be recovered 
by rinsing out the filter flask C. When the apparatus is 
i ready, about 1 gram dry 

sodium carbonate is 
added to the liquid in 
flask A, the stopper 
quickly put in place, 
and the suction started. 

Fig. 2. — Apparatus for the Determination ^vj.1 t 4.1 

or Ammonia According to Shaffer. " ltn a gOOCi pump tUe 

(After Shaffer.) pressure will be re- 

duced to about 10 mm. Hg. in two or three minutes, when, 
the liquid surrounding A being at 50° C, a rapid boiling 
will begin. The temperature is maintained, and the boiling 
allowed to continue for fifteen minutes. At the end of that 
time the ammonia will in all cases have been completely 
given off, and the operation may be stopped by slowly let- 
ting in air at the stop-cock in tube a. The acid in B and B' 
is titrated and the ammonia calculated. One per cent, aque- 
ous alizarin red is used as the indicator. (For a descrip- 
tion of the end-point and the calculation see the preceding 
method of Folin.) 

This method is very accurate, and consumes very little 
time. Shaffer found the results in all cases correct within 
less than 10 mg. of ammonia per liter. Where the neces- 
sary apparatus is available, tins method or that of Folin 
is to be preferred. 

(3) The Formalin Titration Method.— This method was 



THE URINE 17 

first introduced by Bonchese. 1 It is more accurate than 
the Schlosing method, and possesses an advantage over all 
other methods in the rapidity with which a determination 
may be completed. A number of observers have found it 
a satisfactory clinical method for following the ammonia 
excretion in acidosis, but, in its present form, it is not suf- 
ficiently accurate for metabolism experiments, since the re- 
sults are apt to be too high, owing, in part at least, to the 
fact that aminoacids are determined with the ammonia. 
The principle of the method is based on an observation of 
Delepine, and is as follows : The addition of formalin to a 
solution of an ammonium salt gives rise to the formation of 
hexamethylenamin, with the liberation from the salt of the 
corresponding acid. If, then, the acid equivalents in the 
urine be first neutralized with alkali and formalin subse- 
quently added, titration with tenth normal alkali will now 
reveal the acidity due to ammonium salts, and the quan- 
tity of alkali used indicates the amount of ammonia in the 
urine. 

(a) Method of Ronchese. 1 — Ten c. c. of the twenty-four- 
hour specimen of urine are measured with a pipette and 
placed in an Erlenmeyer flask of 300 c. c. capacity. To the 
urine add 100 c. c. of distilled water, previously boiled to 
drive off carbon dioxid, and then 1 or 2 drops of 0.5 per 
cent, alcoholic phenolphthalein solution. Under constant 
stirring add tenth normal sodium hydrate from a burette 
until a pale rose color makes its appearance throughout the 
fluid. Now add 20 c. c. of 20 per cent, formalin (commercial 
formalin is 40 per cent, strength), which has been neutral- 
ized, if necessary, with phenolphthalein as indicator, and 
again add tenth normal alkali, till the same color reaction 

Konchese, A. "Nouveati procede de dosage de 1 'ammoniaque. " Jmr. 
de Pharm et de CHm., 1907, XXV (6th series), 611. 



18 AMMONIA 

is obtained. This is the end-point. To the quantity of 
tenth normal alkali nsed after the addition of the formalin 
a correction of 0.1 c. c. is added for each 3 c. c. required in 
the titration. This sum equals the ammonia content of 10 
c. c. of urine expressed in c. c. of tenth normal ammonia. 
One c. c. of tenth normal ammonia contains 0.0017 gm. am- 
monia. The quantity of ammonia in the twenty-four-hour 
specimen is calculated. 

(b) Method of Bjorn-Andersen and Lauritzen} — Twenty 
c. c. of urine, 5 drops of 0.5 per cent, alcoholic solution of 
phenolphthalein, and 20 gm. of finely powdered neutral 
potassium oxalate are placed in an Erlenmeyer flask, shak- 
en vigorously about one minute, and then titrated imme- 
diately with tenth normal sodium hydrate under constant 
stirring, till a pale rose color is obtained. Now add 5 c. c. 
of commercial formalin (neutralized, if necessary), and the 
acid liberated will cause the color to disappear. Again 
titrate with tenth normal alkali to a pale rose color. (Add 
a little more formalin. If the color remains, the end-point 
has been reached; if it disappears, continue the titration 
until the color no longer fades on the further addition of 
a small quantity of formalin.) The quantity of alkali used 
to obtain the end-reaction after the addition of the for- 
malin represents the number of c. c. of tenth normal ammo- 
nia in 20 c. c. of urine. Each c. c. of tenth normal ammo- 
nia contains 0.0017 gm. of ammonia. 

The entire amount of tenth normal sodium hydrate used 
gives the "total acidity" of the urine. The authors find 
that the curves of total acidity and ammonia run parallel 
in health and in diabetic acidosis. 

1 Bjorn-Andersen, B., and Lauritzen, M. "Ueber Saure- und Ammoniak- 
bestimmung im Harn und ihre klinische Anwendung. ' ; ZeitscJir. f. physiol. 
Chem., 1910, LXIV, 21. 



THE URINE 19 

The quantity of ammonia found is somewhat high in the 
presence of aminoacids. 

(4) Shaffer's Modification 1 of Schlosing-'s Method.— 

This method, which requires at least two days for its com- 
pletion, is more exact than Schlosing's, less accurate than 
Form's, but for most purposes it meets the needs of the 
clinician. An advantage is the simple apparatus required. 
The ammonia, liberated under a bell jar or in a dessicator 
by the addition of stronger alkali, is neutralized by tenth 
normal acid. With a pipette, 25 c. c. of urine are placed 
in the bottom of a dessicator or dish having a diameter of 
15 to 17 cm. An excess of sodium chlorid is added to pre- 
vent decomposition, then about 0.5 gm. of anhydrous so- 
dium carbonate. A second smaller dish containing 20 c. c. 
of tenth normal sulphuric acid is placed in the dessicator or 
under a bell jar with the urine. It is essential that the dish 
containing the urine have a perfectly flat bottom and that 
the depth of the liquid be not more than two mm. "For the 
same amount of urine, the wider the dish the more rapid 
will be the expulsion of the ammonia" (Shaffer). The 
length of the operation may be reduced to forty-eight hours 
by letting the apparatus stand at 38° C. On longer stand- 
ing at this temperature, the ammonia from decomposition 
becomes so great that the results are too high. At room 
temperature (about 25° C.) the apparatus is allowed to re- 
main four days. The acid is then titrated with tenth nor- 
mal sodium hydrate, using alizarin red (1 per cent, aqueous 
solution) as the indicator in the proportion of two drops to 
200 to 300 c. c. of fluid, with a red color, not a violet, as the 
end-point. The ammonia in grams in 25 c. c. of urine is 
found by multiplying 0.0017 by the number of c. c. of JL 
acid neutralized. 

1 Shaffer, P. Loc. cit. (p. 15). 



20 NITROGEN 

NITROGEN 

The total nitrogen of the nrine of a normal adult on a 
mixed diet lies usually between 10 and 16 gm., or about 0.2 
gm. per kilo of body weight. 

Kjeldahl's Method for Determination of Total Nitrogen. 

Eeagents : x 

Crystalline copper sulphate. 
Crystalline potassium sulphate. 
Concentrated sulphuric acid. 
40 per cent, solution of sodium hydrate. 
Talc powder. 

-^.sulphuric acid; n sodium hydrate. 
One per cent, aqueous solution of alizarin red, or 
tincture of cochineal. 

Method. — With a pipette 5 c. c. of the twenty-four-hour 
specimen of urine are measured into a Kjeldahl oxidizing 
flask (Jena glass) of about 800 c. c. capacity. Then add 
about 15 c. c. of concentrated sulphuric acid and about 0.2 
gm. of copper sulphate crystals, and, finally, about 10 gm. 
of potassium sulphate. The flask is placed under a hood 2 
and is heated over a Bunsen burner, 3 with a low flame at 
first, until the foaming has ceased. The heating is con- 
tinued till the contents of the flask become clear. It may be 

1 To determine whether the reagents are N-f ree, proceed with the method, 
substituting 5 c. c. of glucose solution for the urine. If nitrogen is found, the 
necessary correction is evident. 

2 A lead pipe, perforated with holes to receive the necks of the digesting 
flasks (see Fig. 3) and connecting with a flue, is better than most hoods. 
If there is a good draught, the fumes are carried off perfectly. An outlet 
constructed of tile pipes is inexpensive and satisfactory. 

3 The most satisfactory form of apparatus is that designed by Folin and 
made by the International Instrument Co., Cambridge, Mass. The small 
model is shown in Fig. 3. 



THE URINE 



21 



necessary to remove the flask, so that all the charred mat- 
ter may be brought into the acid. After the fluid in the 
flask has become pale green or colorless, the heating is 
prolonged fifteen minutes to insure complete oxidation. 
All of the nitrogenous compounds have been converted to 




Fig. 3. — Digesting Rack for the Kjeldahl Nitrogen Determination. The 
necks of the flasks extend into a perforated lead pipe, which is connected with a 
tile outlet. 



ammonia, which unites with the sulphuric acid to form 
ammonium sulphate. The liquid is allowed to cool (it may 
crystallize eventually) ; about 300 c. c. of distilled water are 
then added, and, when solution is obtained, a heaping tea- 
spoonful of talc powder is placed in the flask (to prevent 
bumping during the boiling). Finally, sufficient 40 per 
cent, sodium hydrate is added to render the solution 



22 



NITROGEN 



strongly alkaline; the quantity required mnst have been 
determined previously. It is well to incline the flask and 
pour the alkali down the side, to prevent mixing and pos- 
sible loss : ammonia. 1 The flask is immediately connected 
with a distilling apparatus (Fig. 4) provided with a Hop- 



/ 


! 'Vwih ft 


I 


iHP 


rl 


T T T-T.-T.Hl 


n i uu 


'ill KM; 



F:c-. ±.— D:=- 



^z Kr. 



asl \~ttt.::^v Dztz~.vtvatt:v. 



kins bulb or similar device to prevent alkali passing over 
into the acid, and its contents boiled. The distillate, con- 
taining the ammonia liberated by the stronger alkali, is 
received in an Erlenmeyer flask in which 25 to 50 c. c. of 
tenth normal sulphuric acid have been placed. The distil- 

2 To gnard against such loss, a doubly perforated stopper may be pro- 
vided and the alkali introduced into the flask by a funnel, whose stem, pass- 
ing through the stopper, is plugged immediately after adding the alkali. 



THE URINE 23 

lation is continued till the distillate is no longer alkaline to 
litmus — usually a half hour or less. The condensing tube 
is then washed with distilled water into the distillate. To 
determine the excess of acid, the contents of the flask are 
titrated with tenth normal alkali with alizarin red (2 drops 
to 200 to 300 c. c. of fluid) as the indicator. The end-point 
is a red color, not a violet. (Tincture of cochineal 1 is used 
at times as an indicator. It imparts a very pale brown 
color to acid solutions when added in the proportion of 
about four drops to 300 c. c. ; when the reaction becomes al- 
kaline the color changes to amethyst.) Since one c. c. of 
tenth normal acid is equivalent to 0.0014 gm. of nitrogen, 
the amount of the latter in 5 c. c. of urine and, finally, in the 
twenty-four-hour specimen is easily computed. 

If insufficient acid has been taken to receive the distil- 
late, the excess of ammonia may be titrated with tenth nor- 
mal sulphuric acid (Hoppe-Seyler, Thierfelder). 

Instead of distilling the oxidized material, the ammonia 
determination may be performed according to Folin's 
method. The titration and calculation of the result are 
performed as described above. Sodium hydrate is substi- 
tuted for sodium carbonate. 

CHLORIDS 

In health, with the usual mixed diet, the chlorids of the 
urine usually amount to 10 to 15 gm. daily. The limits of 
the normal are said to be 6 and 22 gm. 

Qualitative Test 

About 10 c. c. of urine, placed in a test tube, are acidi- 
fied with strong nitric acid, and then one or two drops of 

1 Tincture of cochineal is prepared by grinding cochineal bugs in 50 per 
cent, alcohol in a mortar, allowing the mixture to digest a day, and filtering. 



24 CHLORIDS 

dilute silver nitrate solution (10 to 15 per cent, aqueous 
solution) are added. A white precipitate denotes the pres- 
ence of chlorids. An approximate idea of the quantity 
of chlorids may be gained. Normally, a dense precipitate 
appears and quickly settles to the bottom of the tube. With 
great reduction in the chlorids only a cloud is seen, with- 
out flocculent precipitate. 

QUANTITATIVE DETERMINATION OF ChLOEIDS 

Harvey's x Modification of Volhard's Method.— This 
method is valuable because of its rapidity without sacri- 
fice of accuracy. As in the original method, the chlorids 
are precipitated by adding an excess of silver nitrate, the 
amount of which is determined by titration with ammonium 
thiocyanate. The chlorids are calculated as sodium chlo- 
rid. Albumin, if present in the urine in appreciable quan- 
tity, must be removed by boiling and the subsequent addi- 
tion of dilute acetic acid, before proceeding to the estima- 
tion of the chlorids. Largely in the author's words, the 
method follows : 

Eeagents : 

(a) A silver nitrate solution containing 29.042 gm. of 
chemically pure, crystalline silver nitrate in one liter of 
distilled water ; 1 c. c. of this solution is equivalent to 0.01 
gm. of sodium chlorid. (The silver solution may be stand- 
ardized against a weighed quantity of dry. chemically pure 
sodium chlorid.) 

(b) A solution of ammonium thiocyanate, 20 c. c. of 
which is equivalent to 10 c. c. of the silver nitrate solution. 
As this salt is very hygroscopic, it cannot be weighed with 
sufficient accuracy to make the solution directly. There- 

1 Harrey, S. C. "The quantitative determination of the chlorids in the 

urine." Arch. Int. Med.. 1910. VI. 12. 



THE URINE 25 

fore, 13 gin. of it are dissolved in one liter of distilled 
water, thus making a concentrated solution, whose strength 
is determined by titration against the silver nitrate solu- 
tion, and the requisite dilution made. This is done in the 
following manner : 10 c. c. of the silver nitrate solution 
are measured with a pipette into a beaker, diluted with 
about 20 c. c. of distilled water, 2 c. c. of the indicator (sol. 
c) added, and the whole titrated with the ammonium thio- 
cyanate solution. If, for example, 12 c. c. of the solution 
are used in the titration and the total volume of the thio- 
cyanate solution is 960 c. c, the volume to which it must be 
diluted with distilled water is determined according to the 
equation 12 :20 : i960 :x, in which x represents the required 
volume. 

(c) The indicator containing nitric acid. To 30 c. c. of 
distilled water add 70 c. c. of nitric acid (sp. gr. 1.2, or 33 
per cent.). Saturate this menstruum with crystalline fer- 
ric ammonium sulphate and filter. 

This indicator is recommended, inasmuch as it substi- 
tutes one solution in place of the two (the ferric indicator 
and the acid), and insures the use of the proper amount of 
the acid. Moreover, it is sufficiently concentrated, so that 
it is necessary to use only 2 c. c, and, therefore, it may 
be kept in a small reagent bottle. The stopper of this bottle 
may be a graduated dropper, which can at the same time 
serve to measure and transfer the indicator. 

Method. — With a pipette transfer 5 c. c. of the twenty- 
four-hour specimen of urine (albumin-free) to a small 
beaker or Erlenmeyer flask, and dilute it with about 20 c. c. 
of distilled water. 1 The chlorids in this solution are now 

1 When the urine is highly colored, add 8 to 10 per cent, solution of 
potassium permanganate a drop at a time, until the red color no longer 
fades rapidly, and the urine has become pale yellow. 



26 SULPHATES 

precipitated by adding 10 c. c. of the silver nitrate solu- 
tion with a pipette. Next, place about 2 c. c. of the indica- 
tor in the mixture. The ammonium thiocyanate solution is 
then run in from a burette under constant stirring, until 
the first trace of red shows throughout the mixture. On al- 
lowing the precipitate to settle, the color may easily be 
recognized in the supernatant fluid. If, however, the mix- 
ture is stirred violently, the color will disappear. "When 
the end-point appears on the addition of the first drop of 
ammonium thiocyanate solution (i. e., when the original 10 
c. c. of silver solution is insufficient to precipitate all the 
chlorid), then 10 c. c. more of the silver nitrate solution are 
added, and the titration completed with corresponding al- 
lowance in the calculation. 

The calculation may be made as follows : As 20 c. c. of 
the ammonium thiocyanate solution are equivalent to 10 
c. c. of the silver nitrate solution, divide the number of c. c. 
of thiocyanate solution used by two (2) and subtract the 
quotient from 10 c. c, the amount of silver nitrate origi- 
nally taken. The result is the number of c. c. of silver ni- 
trate solution actually used in the precipitation of the chlo- 
rids. As 1 c. c. of the silver solution is equivalent to 0.01 
gm. of sodium chlorid, the number of cubic centimeters of 
silver nitrate solution used, multiplied by 0.01, will give the 
amount of the chlorids, expressed in terms of sodium chlo- 
rid, in 5 c. c. of urine, the quantity taken. From this the 
total amount of chlorid in the twenty-four-hours specimen 
is calculated. 

SULPHATES 

Quantitative estimation of the sulphates in the urine is 
of no practical value in general clinical work at the pres- 
ent time. Of the sulphates present in the urine, indoxyl 
sulphate alone is tested for in the usual examination. 



THE URINE 27 

The tests for indoxyl sulphate depend on the oxidation 
of indoxyl to indigo blue and its extraction in chloroform. 
It is necessary at times to precipitate the urine, before 
testing, with one-fifth volume of 20 per cent, lead acetate 
to remove pigments, which may interfere with the recogni- 
tion of the blue color. 

Obermayer's Test.— Equal parts of urine and Ober- 
mayer's reagent (0.2 per cent, ferric chlorid in concen- 
trated hydrochloric acid) are mixed in a test tube and al- 
lowed to stand a few minutes (2-3). A small amount of 
chloroform is added, and the test tube is inverted several 
times. With normal amounts of indoxyl sulphate a faint 
blue is seen in the chloroform ; an excess causes a dark blue 
color. By using the same quantities of urine, reagent, and 
chloroform, and test tubes of uniform diameter, daily vari- 
ations in the intensity of the reaction may be followed. 

Jaffe's Test.— Equal quantities of urine and strong hy- 
drochloric acid are mixed in a test tube ; about 2 c. c. of 
chloroform and 1 to 3 drops of strong aqueous solution of 
calcium hypochlorite are added. The tube is inverted sev- 
eral times, and the indigo collects in the chloroform, as in 
the preceding test. 

If the patient has been receiving iodin in any form, a 
violet color is imparted to the chloroform in performing 
Obermayer's and Jaffe's tests. To destroy the color pro- 
duced by the iodin and bring out that of indigo blue, if 
present, the chloroform is transferred to a second test tube 
and is shaken with dilute potassium hydroxid; or water and 
a small quantity of strong sodium thiosulphate solution are 
added to the chloroform and the whole shaken. The violet 
is decolorized, leaving the blue. 

Codein, when administered in large doses, is said to 
give a purplish red color to the chloroform. 



28 ALBUMIN 



ALBUMIN 



Normal urine contains albumin in traces too small to be 
detected with the usual tests. 

Before testing for albumin, two conditions must be ful- 
filled: (1) The urine must be perfectly clear, and (2) its 
reaction must be acid. 

(1) If the specimen to be examined is fresh and fairly 
clear, passage through filter paper usually suffices to ren- 
der it transparent and clear. With urines containing abun- 
dant fine precipitates or many bacteria, simple nitration is 
not satisfactory. Such urine should be shaken with Kiesel- 
guhr (infusorial earth) and then passed through a folded 
filter. The meshes of the paper are plugged, so that the 
nitrate is perfectly clear, though it may be necessary to 
return the first few cubic centimeters of the nitrate to the 
filter. Minute quantities of albumin may be removed by 
the nitration with Kieselguhr. 

(2) If alkaline or neutral in reaction, the urine should 
be rendered slightly acid to litmus by the addition of a few 
drops of 3 per cent, acetic acid. 

Of the following qualitative tests it is advisable to use 
at least two in all instances to avoid error. Heller's and 
the heat and acetic acid tests form a satisfactory combina- 
tion. 

Qualitative Tests 

(1) Heat and Acetic Acid Test.— (a) Fiest Method. — 
A test tube (18 to 20 mm. in diam.) is nearly filled with the 
clear, acid urine. Holding the tube by its lower end, the 
urine in the upper part is boiled over a Bunsen burner or 
spirit lamp, the cool urine in the lower part of the tube 
serving for comparison with the boiled portion. A cloud 



THE URINE 29 

may appear on boiling, due (1) to precipitation of calcium 
phosphate, or (2) to albumin, or (3) to the precipitation of 
both simultaneously. A few drops of 3 per cent, acetic 
acid are now added. If the precipitate be due to phosphates 
alone, it will disappear on the addition of the acid, whereas 
the albumin coagulum will usually be intensified, never les- 
sened, unless a considerable excess of acid is added. When 
both phosphates and albumin are precipitated together, the 
cloud may be perceptibly diminished but not abolished by 
acidification. Very small quantities of albumin may give 
no cloud on heating, but the albumin may appear after the 
addition of the acid. Such traces of albumin are best de- 
tected by holding the tube against a dark background with 
the eye at a right angle to the source of light, for the faint 
cloud may be easily overlooked on casual inspection. The 
urine in the upper part of the tube (which has been boiled) 
is compared with the clear urine in the lower part of the 
test tube. When the urine is of very low specific gravity 
and, therefore, poor in salts, the test is improved by the 
addition of one-fifth to one-tenth volume of saturated so- 
dium chlorid solution to the urine. The urine is not to be 
boiled after the addition of the acid. 

The test is said to indicate albumin in a dilution of 1: 
130,000 (Glaesgen). 

Sources of Error. — (a) There is danger in adding too 
much acetic acid, since the albumin may be converted into 
the soluble acid albumin or syntonin. That it requires a con- 
siderable excess of acid to redissolve the precipitate, how- 
ever, once it is formed, is easily demonstrated. It is help- 
ful to the worker to experiment with known specimens to 
determine the degree of latitude one can safely follow in 
the addition of the acid, (b) Nucleoalbumin may be pre- 
cipitated by heat and acetic acid; it is also thrown out of 



30 ALBUMIX 

solution by the addition of dilute acetic acid to the cold 
urine. Two tests may be performed, one on the cold, the 
other on the boiled, urine: by comparison it is usually \ s- 
sible to estimate whether part or all of the precipitate is 
:lue to the nucleoproteid. Or, the urine is treated with di- 
lute acetic acid, filtered to remove the precipitate of nucleo- 
albumin. a few more drops of the dilute acid added, and the 
contents of the test tube boiled; a precipitate appearing 
now is albumin. The test for nucleoalbumin is improved 
if the urine be diluted with water ; that for albumin is 
sharper after the addition of salt, (c) Following the ad- 
ministration of cubebs. copaiba, turpentine, etc.. resin* 
bodies appear in the urine, and may be precipitated. 
After cooling the fluid the precipitate may be dissolved 
in petroleum benzine or in alcohol, albumin being insoluble. 
id) Albumoses appear after the urine becomes cool: 
the precipitate redissolves on boiling, (e) The Bence- 
Jones' body is coagulated at about 60 c C. but usually 
redissolves in part or wholly as the boiling point is 
reached. 

(b) Second Method. — This method, widely used in 
France, has recently been carefully examined and recom- 
mended by Grlaesgen. 1 The acetic acid is added before the 
specimen is boiled. About 20 c. c. of urine and 5 drops of 
20 per cent, acetic acid are mixed in a test tube. The urine 
in the upper part of the tube is boiled i or the mixture may 
be divided between two test tubes, one to be boiled, the 
other to serve as a control). If the acetic acid prod 
a cloud in the cold (nucleoproteid). the specimen is cleared 
by filtration before boiling. The acidification previous to 
boiling prevents a precipitation of phosphates in the major- 

1 Glaesgen. "Zxa Methodik des Xaehweises sehr kleiner pathologiscber 

_ ;•-:-_ :::. Ham." If f &. Wchnschr., 1911, LYlll, 1123. 



THE URINE 31 

ity of instances; if such a precipitate occurs, a few more 
drops of the acid are added to dissolve it. This will not 
cause the solution of a slight albuminous precipitate, pro- 
vided the specimen is not reboiled. With the precautions 
given, the presence of a cloud or precipitate indicates al- 
bumin. (For the detection of a very faint cloud, see the 
first method.) 

By this method Glaesgen finds that albumin may be 
demonstrated in a dilution of 1:180,000. 1 

(2) Heat and Nitric Acid Test.— The method of pro- 
cedure is the same as in the preceding test (first method), 
the urine in the upper part of the test tube being boiled. 
One to four drops of concentrated nitric acid 2 are now 
added. The precipitate, which may form on boiling the 
urine, may be due to albumin or phosphates or to both. 
The phosphate precipitate is dissolved by the addition of 
the acid; in such case a few more drops of nitric acid are 
added, when albumin is precipitated, if present. When 
more than a trace of albumin is present in the urine, the 
precipitate is flocculent and whitish or brownish. With a 
urine of low specific gravity the addition of one-fifth vol- 
ume of saturated sodium chlorid solution at times makes it 
possible to recognize a trace of albumin, which would oth- 
erwise be missed. The urine may remain clear after boil- 
ing, but a precipitate of albumin may still appear on acidi- 
fication, as in the heat and acetic acid test. If the cloud 
is faint, there is danger of missing it, unless the tube be 
held against a dark background with the eye at a right 
angle to the source of light. Do not boil after adding the 
acid. 

1 A somewhat limited experience with the second method has shown it to 
be quite as sensitive as the first, in the writer 's hands. 

2 Nitric acid becomes yellow from the formation in it of nitrous acid. 
It is readily cleared by the addition of crystals of urea. 



32 ALBUMIN 

The test is said to be as delicate as the heat and dilute 
acetic acid test. 

Sources of Error. — The possibilities of error are much 
the same as in the heat and acetic acid test, (a) An ex- 
cess of acid is to be avoided, as the precipitate may be dis- 
solved, forming acid albumin. The proportion of acid to 
urine should not exceed about 1:1,000 (Simon), (b) Eesi- 
nous bodies are distinguished as in the preceding test, (c) 
Uric acid may precipitate after standing a few minutes. 
The precipitate is crystalline, and gives the murexid test, 
(d) Albumose is soluble in the boiling solution, but in- 
soluble in the cold. The precipitate which forms may be 
redissolved by heating, (e) Bence-Jones' protein usually 
exhibits maximal precipitation at about 60° C, with par- 
tial or complete disappearance of the coagulum at the boil- 
ing point, (f ) In markedly icteric urine a green precipitate 
of biliverdin may be produced. This, unlike coagulated 
albumin, is soluble in alcohol. Finally, it may be added, the 
nitric acid possesses an advantage over dilute acetic acid, 
since its addition to boiling urine does not precipitate mu- 
cin or nucleoproteid. The nitric acid must be free from 
nitrous acid. 1 

(3) Heller's Test.— In performing this test a wide test 
tube or, better still, a conical glass or horismascope should 
be used. Ten to 20 c. c. of urine are placed in a conical 
glass, and then, with the glass inclined, concentrated nitric 
acid 2 is poured slowly down its side. Being the denser 
fluid, the acid collects at the bottom. The glass is now 
brought to the vertical position very gradually, to prevent 
mixing of the urine and acid. If albumin is present in the 
urine, a white precipitate is formed at the line of contact 

1 This 1 refers to footnote 2 on p. 31, beginning li Nitric Acid." 
2 See footnote on p. 31. 



THE URINE 33 

between urine and acid. The precipitate is acid albumin, 
which is insoluble in the great excess of acid. The breadth 
and sharpness of the ring will depend upon the quantity of 
albumin present, and also upon the success with which the 
urine and acid have been layered. When small quantities 
of albumin are present the ring may appear only after two 
or three minutes, and then may be overlooked unless the 
tube is examined against a dark background with the eye 
at a right angle to the source of light. 

Glaesgen 1 finds the reaction positive with albumin in a 
dilution of 1 :35,000. 

Sources of Error. — (a) Urines which have been pre- 
served with thymol may give a ring at the line of contact 
which is practically indistinguishable macroscopically from 
that produced by albumin. 2 Below the ring there is a 
greenish zone extending into the acid, above it a red zone. 
When thymol and albumin coexist, it may be noted that 
the thymol ring forms just beneath that of albumin. The 
thymol may be removed by shaking the urine with an equal 
volume of petroleum ether for two or three minutes, (b) 
Urates may be precipitated, but the ring is y 2 to 1 cm. 
above the line of contact. The ring is broader than that 
caused by albumin, and disappears on warming the urine, 
(c) Nucleoalbumin may produce a ring % to 1 cm. above 
the line of contact. As nucleoalbumin is insoluble in strong 
acid, the ring rises as the acid diffuses upward in the urine. 
The ring is more marked if the urine be diluted with about 
three parts of water, (d) Eesinous acids may form a ring 
above the line of contact. The ring is partially cleared on 
heating. The precipitate, if due to resins, may be pipetted 
off and dissolved in ether. When resinous bodies are sus- 

1 Loc. cit. 

2 Weinberger, W. ' ' Thymol as a source of error in Heller 's test for 
urinary protein. 7 - 1 Jour. A. M. A., 1909, LII, 1310. 



34 ALBUMIN 

pected the following test may be employed: To 8 to 10 
c. c. of urine add 2 to 3 drops of strong hydrochloric acid; 
the resinous bodies are precipitated. Bender strongly acid 
with hydrochlorid acid and heat; a red color develops, (e) 
Albumose and Bence-Jones' body form a ring at the line 
of contact, which disappears more or less completely on 
heating, (f) Urea nitrate may be deposited between the 
fluids. It is easily recognized, as it is not compact and uni- 
form, but manifestly crystalline. Dilution of the urine 
causes its disappearance. 

(4) Potassium Ferrocyanide and Acetic Acid Test.— 
To 10 to 15 c. c. of urine in a test tube add a few drops 
(about 5) of strong acetic acid to render the urine markedly 
acid. Xucleoalbumin, if present, is precipitated and should 
be removed by nitration. Xow add a few drops of 5 per 
cent, potassium ferrocyanide. A cloud or a flocculent pre- 
cipitate indicates albumin. Care must be exercised not to 
add an excess of the ferrocyanide, as the albuminous co- 
aguluni may be redissolved. The test is positive with al- 
bumin in a dilution of 1:70,000 (Glaesgen), but, like the 
preceding tests, its delicacy depends much on the concen- 
tration of the urine in salts. 

Sources of Error. — Albumoses and Bence-Jones' pro- 
tein are coagiilated. but the coagulum disappears on heat- 
ing — completely in the case of albumose, partially with 
Bence-Jones' body. 

Xumerous other tests for the recognition of albumin 
in the urine have been devised. Some of them, as Spieg- 
ler's, are too delicate. The tests given above have been 
thoroughly tested, and are almost universally employed by 
clinicians. Thorough familiarity with them should be suf- 
ficient for all practical purposes. 



THE URINE 35 



Quantitative Determination of Albumin 



Tsuchiya's Modification of the Esbach Method. 1 — 

Tsuchiya has devised a new reagent for precipitating the 
coagulable protein, to be used with the Esbach tube. The 
formula of Tsuchiya's reagent is: 

Phosphotungstic acid 1.5 gm. 

Hydrochloric acid, cone 5.0 c. c. 

Alcohol, 96 per cent., to 100.0 c. c. 

Method. — If alkaline, the urine is acidified with a few 
drops of acetic acid to prevent bubbling, when the reagent 
is added. The Esbach tube is rilled with urine to the mark 
U, and then the reagent is added to the mark R. The tube 
is corked and inverted twelve times to insure thorough and 
uniform mixing of the urine and reagent. (Do not shake, 
since bubbles clinging to the precipitate cause it to float.) 
The tube is placed in a vertical position for twenty-four 
hours at room temperature to allow the precipitate to 
settle, when the height of the precipitate is read on the 
scale marked on the tube. The figure obtained gives the 
quantity of albumin in grams per liter. 

With large quantities of albumin the urine should be 
diluted with water, so that the reading will be below 4 gm. 
per liter, for Mattice has shown that above this the error 
increases greatly. 

Tsuchiya's is a great improvement on the Esbach re- 
agent, and should supplant it. With Esbach 's reagent as 
the precipitant, the results are often not even approxi- 
mately correct. Some of the advantages of Tsuchiya 's re- 
agent over that of Esbach are: (1) that the precipitate 

1 Mattice, A. F. " The quantitative estimation of albumin in the urine. ' ' 
Arch., Int. Med., 1910, V, 313. 



BEXCE-JOXES" BODY 

rarely floats, but _ settles evenly in the bottom of the 
tube : 3 the readings are mneh less affected by slight vari- 
ations in temperature ; 4 the average error is very 
greatly reduced, amounting to less than 0.3 gnu per litei 
(controlled by the Kjeldahl and gravimetric methods), so 
that daily variations in albumin output can be followed 
with considerable accuracy, and (5) the reagent is clean, 
and doe- not stain hands or clothe- Mattice}. Glucose 
in the urine does not interfere with the accuracy of the 
test 

rmal urines usually yield a slight precipitate when 
treated with Tsuchiya - at, but the bulk of it is so 

small that it is not measurable, and in no way interfe 
st 
Removal of Albumin from the Urine.— As albumin in- 
58 with certain reactions, it is necessary at time- tc 
remove it before performing other tests. A convenient 
method is the heat and dilute acetic acid test. The coagu- 
late in is removed by filtration, and the filtrat- teg 
by one of the other tests I _ine whether it is al- 
bumin-! : 

BENCE-JONES' BODY 

This protein is : : ai e eurrence. There is no simple. 

isive. qualitative test which it may be recognized 
Its presence may be strongly suspected, though not abso- 
lutely | by the following reactions : If alkaline or 
neutral, acidify the urine with dilute acetic acid; filter the 
specimen, if necessary, to render it clear. (1) On heating 
the urine slowly in a test tube there ap Bars milky tur- 
bidity at about 52° C; at 60° C. the precipitate is abun- 
dant and sticky. A e temperature rises above 70° C. 



THE URINE 37 

the precipitate usually lessens materially, and may entirely 
disappear at the boiling point, though a slight cloud usu- 
ally persists. As the urine cools the precipitate reappears. 
(2) The addition of an excess of nitric acid to the cold 
urine causes a precipitate, which is partially or completely 
dissolved on boiling, but again separates as the tempera- 
ture becomes lower. (3) Similar reactions may be obtained 
with many of the tests for albumin. That all of these re- 
actions are influenced very greatly by the acidity and the 
salt content of the urine has been shown by Massini. 1 The 
further identification of Bence- Jones' protein is more or 
less complicated ; the reader is referred to the larger works 
or to the literature. 

ALBUMOSE 

The secondary or deuteroalbumoses, though of wide oc- 
currence in the urine in disease, are ordinarily of little 
diagnostic importance. Their presence may be shown in 
the following manner (Simon) : Strongly acidify a few 
c. c. of urine with acetic acid, and then add an equal vol- 
ume of saturated solution of sodium chlorid. The pres- 
ence of albumose is indicated by the occurrence of a pre- 
cipitate, which disappears on boiling and reappears on 
cooling. Since albumin is usually present in the urine 
with albumose, the boiling urine should be filtered to re- 
move the albuminous precipitate. A cloud, which develops 
in the filtrate on cooling, signifies albumose. To the hot 
filtrate an excess of sodium hydrate is added to render it 
strongly alkaline, then 1 per cent, copper sulphate drop by 
drop, when a red color appears (the biuret test). 

1 Massini, E. ' ' Untersuchungen bei einem Falle von Bence- Jones 'scher 
Krankheit." Deutsch. Arch. f. Tclin. Med., 1911, CIV, 29. 



38 GLUCOSE 

GLUCOSE 

(Dextrose, Grape Sugar) 

Glucose is present normally in the nrine in traces, the 
quantity varying between 0.015 and 0.04 per cent, in the 
twenty-four-hour specimen. The amount is so small that 
it is not detected with the usual clinical tests. 

The urine to be tested should be clear. Simple nitration 
may be sufficient. If this fails the urine is shaken with 
powdered normal lead acetate, and then filtered. 

Qualitative Tests 

(1) Trommer's Test.— If more than a trace of albumin 
is present, it should be removed with heat and dilute acetic 
acid. To urine in a test tube add one-third volume of 10 
per cent, sodium or potassium hydrate, then 10 per cent, 
copper sulphate solution — the contents of the tube being 
thorough^ mixed after each addition of the copper — until 
a slight excess of cupric hydroxid remains undissolved. If 
sugar is present in the urine, much more copper sulphate 
can be added before a permanent precipitate is obtained, 
and the percentage of sugar may be roughly estimated in 
this way; the mixture turns deep blue. The upper part of 
the fluid is now heated just to boiling. In the presence of 
glucose cuprous oxid and hydroxid are formed, producing 
in the heated portion greenish-yellow clouds, which gradu- 
ally change to a brick red and diffuse throughout the fluid. 
The rapidity and intensity of the reaction depend upon the 
concentration of the glucose. With a high -percentage of 
sugar, metallic copper may separate as a brownish-red 
coating on the side of the test tube (easily removed with 
nitric acid). 

The test will indicate 0.2 per cent, of dextrose. It 
should always be confirmed by other tests. 



THE URINE 39 

Sources of Error. — When the reduction of the copper is 
atypical, the interrjretation of the result is in doubt. The 
combined glycuronates, uric acid, creatinin, creatin, are all 
capable of reducing copper to a certain extent. They never 
cause more than a dirty yellow ; the granular, red precipi- 
tate of cuprous oxid is missed, for ammonia, creatinin, etc., 
keep in solution the small amounts of cuprous oxid formed 
in sugar-free urines. With less than 0.2 per cent, of glu- 
cose, a similar result may be obtained, for the sugar itself 
may hold in solution a small quantity of cuprous oxid; on 
cooling the red, granular precipitate may appear. The 
alkaptone bodies may also cause an atypical reduction. 
Other hexoses or pentose may be responsible for the reac- 
tion. Before testing with any copper solution, chloroform 
must be removed from the urine by boiling, as it is a fairly 
strong reducing agent. Urine preserved with formaldehyde 
may likewise give a reduction. 

(2) Fehling's Test. 

Solution (l): 1 

Copper sulphate, cryst 34.65 gm. 

Distilled water to 1,000.0 c. c. 

Solution (2) i 1 

Eochelle salt 173.0 gm. 

Sodium hydrate 125.0 gm. 

Distilled water to 1,000.0 c. c. 

More than a trace of albumin should be removed from 
the urine before testing. Equal volumes of solutions (1) 

1 If solution (1) is to be used for qualitative worlc only, it is not neces- 
sary to weigh the copper exactly on an analytical balance. In preparing 
solution (2), dissolve the Rochelle salt in hot water, then cool to room tem- 
perature, add the sodium hydrate and make up to one liter. 



40 GLUCOSE 

and (2) are mixed 1 in a test tube and boiled; the deep blue 
fluid should remain perfectly clear. Now (a) add the urine 
in small amount, never exceeding one-half the volume of 
the mixed solutions originally taken. A yellow or red pre- 
cipitate appears at once. A second way (b) of perform- 
ing the test is to layer the urine over the mixed, boiled so- 
lutions by allowing it to run down the side of the test tube. 
At the line of contact a yellow precipitate, which quickly 
turns red and diffuses downward, is formed in the presence 
of glucose. The precipitate appears within a few seconds. 
With small amounts of glucose, the diffusion downward is 
lost, but the red oxid soon collects at the bottom of the tube. 
With the second procedure (b) there is less likelihood of 
confusion in interpreting the test. 

The test is said to reveal 0.08 per cent, of glucose. 

Sources of Error. — A dirty, greenish-yellow precipitate 
does not mean sugar in the majority of instances. The test 
contains all the sources of error of Trommer's test (q. v.). 
Chloroform, when used to preserve the urine, must be 
driven off by boiling. A precipitate which appears on 
standing means nothing. 

(3) Almen-Nylander's Test.— Eeagent. Four grams of 
Eochelle salt are dissolved in 100 c. c. of warm 10 per cent, 
sodium hydrate. The mixture is saturated with bismuth 
subnitrate (add about 2.0 gm. of the latter), filtered, and 
placed in a dark bottle. The reagent is permanent. 

Albumin must be removed from the urine, since the sul- 
phid of bismuth, which may result from its presence, is 
brown and interferes with the test. 

To the urine in a test tube add one-tenth volume of the 
reagent, mix, and place the tube in a boiling water bath 

1 A mixture of the two solutions is not permanent, and should, there- 
fore, always be freshly prepared at the time of performing the test. 



THE URINE 41 

for five minutes. 1 More prolonged boiling should be 
avoided, otherwise sngar-free urine may reduce the bis- 
muth. If dextrose is present the fluid darkens, and a black 
precipitate of metallic bismuth separates. When the solu- 
tion turns dark only on cooling the test is negative. In a 
sugar-free urine a white precipitate of phosphate is formed. 

The test indicates 0.08 per cent, of glucose, maltose, or 
lactose, and 0.07 per cent, of levulose (Rehfuss and Hawk). 

Sources of Error. — Nylander's solution is not reduced 
by uric acid, creatinin, the alkaptone bodies, pyrocatechin, 
and phosphates, and the test is, therefore, a good control 
of Trommer's and Fehling's tests. Pentose may cause a 
reduction; the same is true of hexoses. The test may be 
positive after eating asparagus, and also after the admin- 
istration of hexamethylenamin (urotropin). An excess of 
combined glycuronates may cause a reduction. Chloroform 
should be removed from the urine by boiling. Formalde- 
hyde, when added to the urine, reduces the bismuth. 

Rehfuss and Hawk agree with Kistermann that any pro- 
tein-free urine which gives a negative Nylander's test may 
safely be said to be sugar-free in a clinical sense. It is 
safer than either of the copper tests, and should be used 
more extensively than it is. 

(4) The Fermentation Test.— When positive, this test 
proves that the reducing body is a fermentable sugar. In 
the vast majority of instances the sugar is glucose. 

A piece of fresh compressed yeast about the size of a 
hazel nut is rubbed in a mortar with about 50 c. c. of urine, 
which is then filled into a fermentation tube, so that the 
air is completely displaced. As controls, use (a) normal 
urine and yeast, and (b) normal urine and yeast plus glu- 

Rehfuss, M. E., and Hawk, P. B. "A study of Nylander's reaction.' ' 
Jour. Biol. Chem., 1909-10, VII, 273. 



42 GLUCOSE 

cose, to prove the activity of the yeast. The three tubes 
are set aside in a warm place (temperature 20° to 37° C.) 
for several hours. If the yeast is active and glucose pres- 
ent, alcohol and carbon dioxid gas will be evolved, the 
bubbles collecting at the top of the tube. No gas, or only a 
minute bubble, should be evolved in the control tube (a), 
whereas the glucose added to control tube (b) should be 
fermented. To lessen the danger of bacterial decomposi- 
tion, the urine may be boiled before testing. The test will 
indicate 0.05 to 0.1 per cent, of glucose. As a further check 
the reduction tests may be repeated with the filtered urine 
after fermentation is completed. 

A positive test indicates the presence of a fermentable 
sugar. 4 

Sources of Error. — Levulose and maltose, if present, 
may be fermented with the evolution of gas. Before add- 
ing the yeast chloroform must be removed from the urine 
by boiling. Thymol and formaldehyde, when used as pre- 
servatives, may inhibit the growth of the yeast. It is said 
that hexamethylenamin in sufficient doses also prevents the 
fermentation. 

(5) Cippolina's x Modification of the Phenylhydrazin 
Test.— Albumin, when present, should be removed before 
performing the test. 

To 4 c. c. of urine in a test tube are added 5 drops of 
pure phenylhydrazin (the base) and 0.5 c. c. of glacial acetic 
acid (or 1.0 c. c. of 50 per cent, acetic acid) ; the mixture 
is boiled gently over a low flame for one minute. Now add 
4 to 5 drops of sodium hydrate (sp. gr. 1.160) ; the mix- 
ture must still remain acid. The whole is heated a few sec- 
onds longer, and set aside to cool. Immediately or within 

1 Cippolina, A. "Ueber den Nachweis von Zucker im Ham." Deutsche 
med. Wchnschr., 1901, XXVII, 334. 



THE URINE 43 

about twenty minutes, especially with a urine of low spe- 
cific gravity, the characteristic sheaf-like yellow needles of 
phenylglucosazone appear. Since their size is subject to 
considerable variation, high magnification is necessary at 
times to see them. 

The test is very delicate, indicating 0.05 per cent, of 
glucose. However, the sensitiveness of the test depends 
very largely upon the specific gravity of the urine. Con- 
centrated urines may react negatively in the presence of 
less than 0.2 per cent, of glucose. The reason for this is 
that phenylglucosazone crystals are held in solution in the 
presence of much urea, ammonium salts, and other nitroge- 
nous bodies. But with more than 0.2 per cent, of glucose 
typical crystals form within a few minutes, regardless of 
the specific gravity of the urine. 

In place of the characteristic needles yellow balls, which 
change to thorn-apple forms or rosettes, may be obtained. 
The latter are seen only in urine containing a pathological 
quantity of sugar, never in a normal urine (Cippolina). 
Characteristic needles arranged in sheaves may be obtained 
by recrystallization from hot 60 per cent, alcohol. When 
the crystals are atypical the specimen should be set aside 
and reexamined at the end of one hour. 

To determine definitely that the crystals are derived 
from glucose and not from another sugar, it is necessary 
to filter them off and purify them by repeated recrystal- 
lization from hot 60 per cent, alcohol. (The melting point 
of the purified crystals may be determined 1 ; that of phenyl- 
glucosazone is 204 to 205° C. The melting point of levu- 
losazone is the same, while maltosazone crystals melt at 

1 For a description of methods, with critical discussion, see Menge, G. A. 
"A study of melting-point determinations." Bull. No. 70, Hyg. Lab., U. S. 
Pub. Health & Mar. Hosp. Serv., Wash., 1910. 



44 GLUCOSE 

about 207° C. The value of melting point determinations 
for the identification of one of the three sugars mentioned 
is, therefore, not great, though very helpful in differentiat- 
ing the osazone of pentose, melting point 168° C, somewhat 
less so with lactose, 200° C.) 

The dry, purified crystals may be identified by dissolv- 
ing 0.2 gm. of them in 4 c. c. of pure pyridin, to which 6 c. c. 
of absolute alcohol are subsequently added, and the whole 
well mixed. The 100 mm. tube of the polariscope is then 
filled with this mixture. Phenylglucosazone gives a levoro- 
tation of — 1° 30'. This procedure is seldom, if ever, neces- 
sary in clinical work. 

Quantitative Estimation of Glucose 

(1) Benedict's * First Method.— This is one of the best 
and quickest quantitative methods for the clinician. The 
solutions required are: 

Solution A: 

Eecrystallized copper sulphate 2 .. 69.3 gm. 
Distilled water to 1,000.0 c. c. 

Solution B: 

Crystalline Kochelle salt 346.0 gm. 

Anhydrous sodium carbonate .... 200.0 gm. 

Distilled water to 1,000.0 c. c. 

Solution C: 

Potassium sulphocyanide 200.0 gm. 

Distilled water to 1,000.0 c. c. 

1 Benedict, S. E. "The detection and estimation of reducing sugars." 
Jour. Biol. Chem., 1907, III, 101; also N. Y. Med. Jour., 1907, LXXXVI, 
497. 

2 This must be accurately weighed on an analytical balance. Sols. B and C 
do not require exact weights of the substances. 



THE URINE 45 

For use, these solutions are mixed in equal proportions 
in the order in which they are given. As the mixed solu- 
tion (which is blue) keeps fairly well, it is practicable to 
prepare 300 c. c. or more, according to the demand. For 
measuring, pipettes or standard flasks are required. 

Thirty c. c. of the mixed solution are transferred with 
a pipette to an evaporating dish. To this are added 2.5 to 
5.0 gm. of anhydrous sodium carbonate, in order to in- 
crease the alkalinity of the fluid. (For titrating dilute 
sugar solutions, the larger quantity of carbonate will be 
required, since the greater amount of urine which must be 
added will diminish the concentration of the alkaline salt.) 
The mixture is now placed over a Bunsen burner and is 
boiled, until the carbonate is dissolved. A small piece of 
washed absorbent cotton is added to prevent bumping. 
Urine is now run into the boiling solution from a burette 
until a heavy chalk-white precipitate of cuprous sulpho- 
cyanide is formed, and the blue color of the fluid begins to 
lessen perceptibly. The remaining portions of urine should 
be added in quantities of from two to ten drops (depending 
on the depth of color remaining and the relative strength 
of the sugar solution), with vigorous boiling of about one 
minute between each addition. The end-point is the com- 
plete disappearance of the blue color. The point is very 
sharp, and may be obtained with a single drop. If the 
precipitate be allowed to settle, the color in the supernatant 
fluid is more easily appreciated. 

For the complete reduction of the copper contained in 
30 c. c. of the mixed solution, 0.073 gm. of glucose are re- 
quired. The quantity of urine used from the burette, there- 
fore, contains 0.073 gm. of glucose. From this value the 
amount of glucose in the twenty-four-hour quantity of urine 
is calculated. 

5 



46 GLUCOSE 

When the urine is highly colored, its addition to the 
mixed solution may leave a yellowish supernatant fluid. 
To avoid this the urine may be decolorized by first shaking 
it with finely powdered normal lead acetate. The filtered 
urine is then almost colorless. 

Urine which has been preserved with chloroform may 
cause a precipitate of the red oxid to form in place of the 
white cuprous sulphocyanide. This difficulty is obviated by 
first boiling the urine to drive off the chloroform. It may 
also be overcome by substituting for solution C the follow- 
ing: 

Solution D: 

Potassium ferrocyanid 30.0 gm. 

Potassium sulphocyanid 125.0 gm. 

Anhydrous sodium carbonate 100.0 gm. 

Distilled water to 1,000.0 c. c. 

(2) Benedict's 1 Second Method.— This method appears 
to be an improvement on Benedict's first method, in that 
the three solutions are replaced by one, which is permanent. 
As in the preceding method, a white precipitate of cuprous 
sulphocyanid is formed. Benedict's directions for the 
preparation of the solution and for the titration follow: 

Crystallized copper sulphate 18.0 gm. 

Anhydrous sodium carbonate 2 . . . 100.0 gm. 

Sodium citrate 200.0 gm. 

Potassium sulphocyanate 125.0 gm. 

Five per cent, potassium ferrocy- 
anid solution 5.0 c. c. 

Distilled water to 1,000.0 c. c. 

1 Benedict, S. E. "A method for the estimation of reducing sugars. ' ' 
Jour. Biol. Chem., 1911, IX, 57. 

2 200.0 gm. of the crystallized salt may be used. 



THE URINE 47 

With the aid of heat dissolve the citrate, carbonate, and 
sulphocyanate in enough water to make about 800 c. c. of 
the mixture, and filter. Dissolve the copper sulphate sep- 
arately in about 100 c. c. of water, and pour the solution 
slowly into the other liquid, with constant stirring. Add 
the ferrocyanid solution cool, and dilute to exactly one 
liter. Of the various constituents, only the copper sulphate 
need be weighed with exactness. Twenty-five c. c. of the 
reagent are reduced by 0.050 gm. of glucose, or by 0.053 
gm. of levulose. 

Method. — With a pipette measure 25 c. c. of the reagent 
into a porcelain evaporating dish (25 to 30 cm. in diam- 
eter) and add 5 to 10 gm. of anhydrous sodium carbonate 
(or twice the weight of the crystallized salt), and a very 
small quantity of powdered pumice stone. Heat the mix- 
ture to vigorous boiling over a free flame till the carbonate 
is dissolved, and from a burette run in the twenty-four-hour 
specimen of urine (diluted accurately 1 :10, unless the sugar 
content is known to be very slight) quite rapidly, until a 
heavy white precipitate is produced, and the blue color of 
the solution begins to diminish perceptibly. From this 
point the urine is run in more and more slowly, with con- 
stant vigorous boiling, until the disappearance of the last 
trace of blue color, which marks the end-point. An interval 
of 30 seconds' vigorous boiling should be allowed between 
each addition of urine. 

The following explanatory points may be added regard- 
ing the solution : When ready mixed, the solution appears 
to keep indefinitely without any special precaution, such as 
exclusion of light, etc. The trace of ferrocyanid is added 
to prevent precipitation of red cuprous oxid, which may be 
caused by certain impurities. Chloroform has such a 
marked tendency in this respect that it must not be pres- 



48 GLUCOSE 

ent during the titration. The additional alkali is added 
prior to the titration in order to provide sufficient alkalin- 
ity to insure a sharp end-point. Should the mixture become 
too concentrated during the titration process, distilled 
water may be added to replace the volume lost by evapora- 
tion. 

(3) Polariscopic Determination.— The polariscope is 
an expensive instrument, and for this reason it is not as 
generally employed for sugar determinations as the rapid- 
ity and ease of its use would seem to warrant. For clinical 
use the instrument is supplied with two specially made 
tubes, 94.7 mm. and 189.4 mm. long, which permit a direct 
percentage reading of glucose ; the short tube is used with 
dark, highly colored urines, the readings obtained being 
divided by two. The tubes must be perfectly clean and 
dry before using; hot water or fluid should not come in 
contact with them, since the expansion of the glass against 
the outer brass tubing may crack the former. 

The twenty-four-hour specimen, acid in reaction, is fil- 
tered and decolorized, if necessary. This is best accom- 
plished by the addition of about 2 gm. of finely powdered 
normal lead acetate to the urine, which is then shaken 
vigorously and filtered. The first cloudy portions of the 
filtrate are returned to the filter, until the filtrate, which 
is almost colorless, is perfectly clear. Practically no sugar 
is held back by the normal lead acetate. 1 The clear urine 
is now filled into the polariscope tube (189.4 mm. in length) 
until the fluid is convex above the end of the tube. The 
glass disc is then placed over the end of the tube and se- 
cured in place by screwing down the metal cap. Air bubbles 

1 Neuberg, C, II. "Ueber Klarung und Entf arbung. " Biochem. Ztschr., 
1910, XXIV, 423. 



THE URINE 49 

must be avoided, since their presence makes a satisfactory 
reading impossible. The tube is now placed in the polari- 
scope, which must be in a dark room. For illumination a 
sodium flame is used. After focusing, readings are made, 
first without the urine, to determine whether the zero point 
is accurate, next, after refocusing, with the tube of urine; 
starting at zero, the handle is rotated until the entire field 
is equally illuminated. At least six readings should be 
made. The percentage is read directly from the scale, 
tenths being obtained on the vernier. (In case the instru- 
ment is supplied only with the standard tubes of 100 and 
200 mm. length, the percentage of glucose may be calcu- 
lated from the polariscopic readings by dividing the re- 
sults by 0.527.) . 

The method gives fairly satisfactory results. When no 
disturbing bodies are present in the urine, the error is 
about 0.1 per cent, of glucose. 

Sources of Error. — (1) Albumin, when present, must be 
removed before making polariscopic determination of glu- 
cose, otherwise the albumin, which is levorotatory, will 
counterbalance the dextrorotatory glucose, in part at least. 
(2) Alkalinity of the urine precludes its use with the po- 
lariscope, since it has been shown that in alkaline media 
dextrose may be converted into levulose. 1 The addition 
of a preservative to the specimen usually suffices to pre- 
vent an acid urine becoming alkaline. (3) (3-oxybutyric 
acid is levorotatory, and its presence, therefore, interferes 
with the accurate estimation of glucose. (4) The combined 
glycuronates are levorotatory, though they are generally 
present in such small quantity as to produce only slight 
rotation of the polarized light. (5) Levulose, when present 

1 Koenigsf eld, H. ' ' Zur Klinik unci Pathogenese der Lavulosurie beim Dia- 
betes mellitus." Ztschr. f. Min. Med., 1910, LXIX, 291. 



?: .tv ; -e 

in the urine with glncose, is antagonistic, and lowers the 
reading for glucose. (6) Maltose is occasionally present in 
the nrine with glncose. Since it is more powerfnlly dextro- 
rotatory than glncose, the reading may give a valne which 
is too high. 

m the foregoing it is apparent that the error arising 
from /S-oxybutyrie acid may be estimated approximately 
by making polariscopie examination of the specimen be- 
fore and after fermentation with yeast. With the combined 
presence of glncose and levnlose. the relative proportions 
of each may be determined with a fair degree of accu: 
by comparison of the value obtained by titration with cop- 
per solution and the polariscopie value. 

(4) Robert's Specific Gravity Method. 1 — This method 
depends upon a lowering of the specific gravity of the 
nrine as a result of fermentation of the sugar. By obtain- 
ing the specific gravity of the fermented and the unfer- 
mented urine, the quantity of sugar may be calculated. 
About 2.0 gm. of compressed yeast are rubbed in a mor- 
tar with 50 c. c. of the twenty-four-hour specimen of urine, 
acidified with acetic acid if necessary. The specific gravity 
of the suspension is taken at once, the temperature of the 
mixture being noted. The mixture is set aside in a warm 
temperatm _ " in a receptacle plugged with 

cotton, or, better, in a large fermentation tube. When fer- 
mentation is complete the yeast settles to the bottom of the 
flask ; it is well, nevertheless, to test the fluid to determine 
the complete disappearance of the sugar. The mixtur- is 
well stirred and a small portion removed. It is filtered 
and tested with Fehling's solution. If glucose still re- 
mains, the fermentation is allowed to continue, until there 

a L©hnstem, Th_ "Ueber die demimetrisehe Bestummmg des Tranben- 
zuckers im Haine." Arch. f. d. ges, FkysioL, 1895-6, T,XTT, 82. 



THE URINE 



51 



is no longer a reduction of the copper. The mixture is 
again stirred thoroughly to restore the suspension, and the 
specific gravity is determined the second time. It is im- 
portant that the temperature of the suspension at the times 
of determining specific gravity does not differ by more 
than 1° C. The usual urino- 
meters are too inaccurate for 
the determination of the spe- 
cific gravity, which should be 
carried to the fourth decimal 
place. Lohnstein's instrument 
(Fig. 5) is convenient and sat- 
isfactory for the purpose. It 
is an areometer, in whose stem 
the pan, A, is mounted. It is 
floated in the urine and 
weights are placed on the pan 
until the shelf, C, is exactly on 
a level with the surface of the 
fluid. The sum of the weights 
on the pan is the specific grav- 
ity of the fluid. If the pan is 
loaded too heavily, so that the 
surface, C, sinks into the fluid, 
it must be removed and dried. 
The quantity of sugar is calculated by multiplying the dif- 
ference in the specific gravities by the factor 234. The re- 
sult is glucose in grams per cent. When unfiltered urine is 
used (i. e., for the second determination, after fermentation 
is completed), the error does not exceed 5 per cent. 
(Lohnstein). The method permits the determination of 
glucose in strengths of 0.1 per cent, or more. 

(5) Measurement of the carbon dioxid gas formed dur- 




Fig. 5. — Lohnstein's Areometer. 
A, pan for weights; C, shelf which 
should be level with the surface 
of the fluid; E, air chamber. 
(After Lohnstein.) 



52 



GLUCOSE 



ing fermentation has been used to determine the dextrose 
content of urine. One of the most widely known forms of 
apparatus for this purpose is Ein- 
horn's. More accurate results have 
been obtained with Lohnstein's appa- 
ratus (Fig. 6). This consists of a J- 
shaped tube mounted on a stand. In 
the short arm of the tube a bulb is 
blown, the outlet of which may be 
closed by a glass stopper. A hole in 
the neck may be brought opposite a 
similar opening in the hollow stopper. 
The long arm is provided with a scale. 
A quantity of mercury and a pipette 
for measuring the urine are supplied 
with the apparatus. 

Method. 1 — The mercury is poured 
into the apparatus. The bulb is partly 
filled, and mercury extends a short 
distance up the long arm of the tube. 
Then, with the pipette. 0.5 c. c. of 
urine and 0.1 to 0.2 c. c. of a yeast 
suspension 2 (1 part of compressed 
yeast to 2 to 3 volumes of water) are 
placed in the bulb on the surface of 
the mercury. The glass stopper 
(greased with vaselin 20 per cent., in 
yellow wax) is placed in the neck of 

1 Lohnstein, T. ' ' Ueber Garungs-Saecharometer 
nebst Besckreibung eines neuen Garungs-Saccha- 
rometers fur unverdiinnte Urine." Munchen. 
med. JVchnschr.. 1899, XLVI, 1671. 
2 The quantity of yeast suspension employed depends upon the concentra- 
tion of glucose. With very low percentage of sugar the yeast may be rubbed 




Fig. 6. — Lohnstein's Fer- 
mentation Saccharo- 
meter for undiluted 
Urine. (After Wood.) 



UP 



with 10 to 15 volumes of water. 



THE URINE 53 

the bulb in such a way that the openings are opposite one 
another; this is to avoid a positive pressure on inserting 
the stopper. The scale is now placed on the long arm of 
the tube; the zero line should correspond with the level 
of the mercury in the long arm. The stopper is turned, 
to close the bulb, and a weight (provided with the ap- 
paratus) is placed over it to prevent it from being blown 
out, as the carbon dioxid forms. The apparatus is placed 
in an incubator at a temperature of 32 to 38° C. for 
4 to 5 hours, or at room temperature for 24 hours. The 
carbon dioxid formed in the bulb from fermentation of 
the sugar forces the mercury into the long arm of the 
tube. The percentage of glucose is read directly from 
the scale, which is provided with two columns of figures, 
one for the average room temperature, the other for body 
heat. 

LEVULOSE 

Levulose, when present in the urine, is usually asso- 
ciated with dextrose. Occasionally it is the only sugar in 
the urine, a few cases of levulosuria having been reported. 1 
Levulose responds to most of the tests for glucose. It is 
fermentable, reduces copper and bismuth, gives the phenyl- 
hydrazin test; there are, however, certain dissimilarities 
by which the two sugars may be separated. 

(1) Seliwanoff 's Test, as Modified by Borchardt. 2 — Five 
to 10 c. c. of urine and an equal volume of 25 per cent, hy- 
drochloric acid (i. e., 2 parts of concentrated hydrochloric 
acid and one part of water) are mixed, and a few grains 

1 Strouse, S., and Friedman, J. C. il Laevulosuria. ' ' Arch. Int. Med., 
1912, IX, 99. 

2 Borchardt, L. "Ueber die diabetische Lavulosurie und den qualitativen 
Nachweis der Lavulose im Harn. " Ztschr. f. physiol. Chem., 1908, LV, 241. 



54 LEVULOSE 

of resorcin added. The mixture is boiled gently for a few 
seconds. A red color appears, usually followed by a 
brownish precipitate, if levulose is present. The fluid is 
now cooled, poured into an evaporating dish or beaker, 
and treated with sodium carbonate in substance until the 
reaction of the mixture becomes alkaline. It is then 
returned to a test tube, and shaken with acetic ether 
(ethyl acetate). In the presence of levulose the acetic 
ether is colored yellow. The test indicates 0.05 per cent, 
of levulose. 

Sources of Error. — (1) The simultaneous presence of 
nitrites and indican in considerable quantity may yield a 
positive reaction. The nitrites may be destroyed by acidi- 
fying the urine with acetic acid and boiling for one minute. 
(2) Large quantities of indican alone may interfere with 
the reaction by imparting a blue color to the acetic ether, 
making it impossible to recognize the yellow color from 
levulose. In such case the indican is removed by treating 
the urine with an equal volume of Obermayer's reagent 
and extracting several times with chloroform, until the lat- 
ter is no longer colored blue. The fluid is then poured into 
a fresh test tube, the chloroform being discarded, and is 
diluted with one-third volume of water, in order to reduce 
the strength of the hydrochloric acid to 12 to 13 per cent. 
A knife point of resorcin is now added, and the SeliwanofT 
test carried out. (3) Urorosein, when abundant in the 
urine, may impart a reddish- violet color to the acetic ether. 
To remove the pigment before applying Seliwanoff's test, 
take equal quantities of urine and 25 per cent, hydrochloric 
acid, and extract the mixture two to three times in a sep- 
arating funnel with amyl alcohol. Discard the amyl alco- 
hol, which contains the urorosein, add resorcin, and pro- 
ceed with the test in the usual way. (4) It has been found 



THE URINE 55 

that patients taking santonin or rhubarb may give a posi- 
tive SeliwanofT reaction. Discontinuance of the drug causes 
the reaction to disappear. 

In performing the test prolonged boiling should be 
avoided. Borchardt finds no interference with the reac- 
tion from the presence of glucose, lactose, maltose, arabi- 
nose, or glycuronic acid compounds. Saccharose may yield 
a positive reaction, since boiling it with acid liberates levu- 
lose. 

(2) The phenylhydrazin test (see p. 42) gives crystals 
of phenyllevulosazone. They are indistinguishable micro- 
scopically from phenylglucosazone ; the melting point of 
each is the same. The crystals can be positively identified 
by determining their rotation of polarized light. They are 
purified by repeated crystallization from hot 60 per cent, 
alcohol. Then 0.2 gm. of the pure crystals are dissolved 
in 4 c. c. of pyridin, and 6 c. c. of absolute alcohol are added. 
The mixture is poured into the 100-mm. tube of the polari- 
scope and examined. Levulosazon'e gives a dextrorotation 
of 1° 20'. 

Levulosuria combined with glycosuria should be sus- 
pected when the quantity of glucose found on polari- 
scopic examination falls short of that shown by titration. 
A positive SeliwanofT reaction and the lack of a levo- 
rotatory body after fermentation practically confirm 
it. 

Pure levulosuria offers no difficulties in recognition, if 
access to a polariscope may be had. The levorotation of 
the urine, together with positive SeliwanofT and reduction 
tests and the presence of a fermentable substance, makes 
the identification sufficiently complete, if all tests are nega- 
tive after fermentation. The phenylhydrazin test, as de- 
scribed above, removes all doubt, if positive. Levulosuria 



56 LACTOSE 

is usually unsuspected, unless the urine be examined with 
a polariscope. 

Alimentary levulosuria has been used in hepatic diag- 
nosis. In the absence of derangement of hepatic function 
an individual can take 100 gm. of levulose on a fasting 
stomach without the subsequent appearance of levulose. as 
a general rule. On the other hand, the majority of patients 
with liver disease exhibit levulosuria under such condi- 
tions. 1 

MALTOSE 

Maltose is occasionally present in the urine, usually in 
association with glucose. It reduces copper and bismuth 
solutions, and is fermentable. Maltose has about two and 
one-half times the dextrorotatory power of glucose, where- 
as its reducing power is only about two-thirds that of glu- 
cose. Therefore, its presence may be suspected when po- 
lariscopic values exceed the results found with the reduc- 
tion methods. 

LACTOSE 

Lactose may appear in the urine of women physiologi- 
cally in connection with stasis of milk in the breasts. Cu- 
pric salts are reduced more slowly than by glucose. Ani- 
moniacal silver nitrate is reduced by lactose in the cold. 
Lactose is not fermentable by yeast. If equivocal results 
are obtained with the usual compressed yeast, the fermen- 
tation may have been due to contaminating bacteria. With 
a pure culture of Saccharomyces apiculatus. lactose is not 
fermented. 

1 For a discussion of this test, see Churchman. J. W. "The Strauss test 
for hepatic insufficiency. " ' Bull. Johns Hopkins Hosp., 1912, XXIII, 10. 



THE URINE 57 



SACCHAROSE 



Saccharose is seldom encountered in the urine. It is 
dextrorotator} 7 . After inversion (heat 75 c. c. of the urine 
with 5 c. c. cone. HC1 for five minutes at a temperature be- 
tween 68 and 70° C), the fluid becomes levorotatory, since 
the levulose which is formed more than neutralizes the 
glucose. Keduction tests become positive after inversion 



of the sugar. 



PENTOSE 



Pentose is rarely found in the urine. Pentoses are 
sugars with five carbon atoms. The only one of importance 
in the urine — r-arabinose — is, unlike other sugars, optically 
inactive. It reduces copper and bismuth solutions slowly 
and incompletely; with Nylander's solution a grayish pre- 
cipitate may be obtained. Pentose does not ferment with 
yeast. Pentose should be suspected when the reduction 
tests are atypical, when they persist after attempts at fer- 
mentation, when the urine is inactive on polariscopic exam- 
ination. The following tests may also be employed : 

(1) The Phloroglucin Test.— To about 5 c. c. of urine in 
a test tube add an equal volume of concentrated hydro- 
chloric acid and a liberal knife-point (ca. 30 mg.) of phloro- 
glucin. The mixture is heated, preferably on a water bath. 
A red color appears, and, soon afterward, a dark precipi- 
tate forms. The contents of the test tube are cooled, and 
are then extracted with amyl alcohol. Spectroscopic exam- 
ination of the amyl alcohol extract reveals a band midway 
between D and E, a little to the right of the sodium line. 

Sources of Error. — Glycuronic acid compounds yield a 
positive phloroglucin test, including the absorption band, 
thus lessening greatly the value of the test. Lactose and 



58 PENTOSE 

galactose give the same color reaction as pentose, but the 
characteristic absorption spectrum is lacking. 

(2) The Orcin Test.— Equal parts of the urine and con- 
centrated hydrochloric acid (sp. gr. 1.19) and a small knife- 
point of orcin are boiled gently. If pentose is present a 
dark greenish color soon develops, and, finally, a turbidity, 
due to a dark blue or green precipitate. The contents of 
the test tube are cooled, until they are lukewarm, and are 
then extracted with amyl alcohol. The latter exhibits a 
dark, olive-green color, the depth of which depends largely 
upon the concentration of pentose in the urine. If the fluid 
is cold instead of lukewarm when extracted, the amyl al- 
cohol is reddish and the absorption bands are not so plainly 
visible (Salkowski). Spectroscopic examination reveals a 
band at D, the sodium line. 

Sources of Error. — The orcin test is also given by the 
paired glycuronic acid compounds. However, the latter 
react with orcin less readily than with phloroglucin, so 
that of the two the orcin test is to be preferred. It has 
been shown 1 that filter paper may contain pentose-like sub- 
stances, which are soluble in hydrochloric acid. The urine 
should, therefore, not be passed through filter paper. 
Glass wool or asbestos should be employed in its stead. 

(3) Bial's Modification 2 of the Orcin Test. 

Eeagent : 

Orcin t 1.0 gm. 

30 per cent, hydrochloric acid 500.0 c. c. 

10 per cent, ferric chlorid 25 drops 

Keep the reagent in a dark bottle. 

1 Umber, F. ' ' Notiz liber Pentosenreactionen in filtrirten Fliissigkeiten. ' ' 
Berlin. Jclin. Wchnschr., 1901, XXXVIII, 87. 

2 Bial, M. ' ' Ueber die Diagnose der Pentosurie mit dem von mir angege- 
benen Eeagens. ' ' Deutsche med. Wchnschr., 1903, XXIX, 477. 



THE URINE 59 

Method. — Heat about 4 c. c. of the reagent to boiling 
and then add a few drops of the urine to be tested. With 
pentose a green color develops immediately or in a few 
seconds. The quantity of urine employed should not ex- 
ceed 1 c. c. Performed in this way, the test reacts only 
with pentose, not with paired glycuronic acid compounds 
(Bial). 

The green fluid is extracted with amyl alcohol and ex- 
amined spectroscopically, as in the orcin test. 

The specificity of the test has been questioned by a num- 
ber of observers. 

GLYCURONIC ACID 

Glycuronic acid (glucuronic acid) does not appear as 
such in the urine, but becomes paired or conjugated in the 
body with various substances, such as indoxyl, skatoxyl, 
phenol, in which form it is excreted in the urine. In small 
quantity it is normally met with. Glycuronic acid also 
combines with numerous drugs. Urochloralic acid, the 
chloral hydrate compound, is an example. When present 
in considerable amount, the glycuronates may lead to diffi- 
culty in analysis, since many of their reactions resemble 
those of glucose and other carbohydrates. 

With copper solutions the glycuronic acid compounds 
may give a slow, atypical reduction, often a greenish-yel- 
low precipitate — a reaction quite like that produced by pen- 
tose or by very weak solutions of dextrose. The Nylander 
test may be positive. The phloroglucin and orcin tests are 
given by the combined glycuronates. The glycuronates do 
not give the phenylhydrazin test of Cippolina, though the 
test may become positive if the urine be boiled previously 
with 1 per cent, sulphuric acid to liberate glycuronic acid; 



60 GLYCURONIC ACID 

the crystals obtained melt at 114° to 115° C. The combined 
glycnronates do not ferment with yeast. It happens, there- 
fore, that, when the urine contains abnormal quantities of 
both glucose and glycuronates, fermentation does not cause 
a complete loss of reducing power. The glycuronic acid 
compounds are levorotatory in acid urine (inactive if the 
reaction is alkaline), whereas glycuronic acid itself is dex- 
trorotatory. Therefore, boiling one to five minutes with 
1 per cent, sulphuric acid changes a levorotation to dextro- 
rotation, or, if glucose be present, it may increase the dex- 
trorotation, provided some of the sugar is not destroyed. 
Pentose (r-arabinose) is optically inactive. /?-oxybutyric 
acid is levorotatory. To distinguish between the levorota- 
tion produced by this acid and that due to the combined 
glycuronates, the urine is precipitated with subacetate of 
lead and filtered. The glycuronates are precipitated, while 
/?-oxybutyric acid appears in the filtrate, where its presence 
may be indicated by polariscopic examination. Or, the fi- 
oxybutyric acid may be extracted by shaking the urine with 
ether three or four times, the glycuronates remaining in 
the urine. 

Normal urine may contain enough levorotatory sub- 
stances to produce 0.1 degree of levorotation; when the 
glycuronates are increased, the levorotation is 0.2 degree 
or more. 

B. Tollens' Test. 1 — To 5 c. c. of urine in a test tube add 
a bit of naphthoresorcin about the size of a millet seed and 
then 5 c. c. of concentrated hydrochloric acid (sp. gr. 1.19). 
Boil the mixture gently about one minute, and set the tube 
aside for about four minutes. Now cool the contents of the 

1 Tollens, C. ' ' Ueber den Glykuronsauren Nachweis durch die B. Tollensche 
Eeaktion mit Naphthoresorcin und Saizsaure. ' ' Munchen. med. Wchnshr., 1909, 
LVI, 652. 



THE URINE • 61 

tube under running water. Extract with an equal volume 
of ether. (The separation of the ether may be hastened by 
the addition of a few drops of alcohol.) If glycuronates 
are present in the urine in excess, the ether extract is dark 
blue to violet, while with smaller amounts a faint bluish 
or reddish violet color is obtained. Examined spectroscopi- 
cally, the ether extract shows a single dark band near 
the sodium line. (The examination should be made at 
once, 1 as the substance giving rise to the band is not 
stable.) In place of naphthoresorcin in substance, 0.5 c. c. 
of a 1 per cent, alcoholic solution of naphthoresorcin may 
be substituted. The test is sufficiently delicate to detect 
the small quantities of glycuronates present in normal 
urine. 

The dark pigments formed in this reaction by pentoses 
and other sugars are insoluble in ether. 



ALKAPTONURIA 

Alkaptonuria is a very rare condition, a disturbance in 
metabolism. Of the alkapton bodies, two, homogentisinic 
acid and uroleucinic acid, have been isolated. When pres- 
ent in the urine they may give to it the following charac- 
teristics : The fresh urine is markedly acid. It is normal 
in color when voided, but on standing oxidation quickly 
changes the color to a reddish-brown and, finally, to a black. 
The color changes occur more rapidly when the reaction of 
the urine is alkaline. The urine reduces copper and silver 
(the latter in the cold) but not bismuth. The urine does 
not give the phenylhydrazin test, does not rotate the plane 
of polarized light, and is not fermentable. 

1 Brooks, B. Personal communication. 



62 ACETONE 



ACETONE 



Acetone, a ketone, occurs in normal urine in amounts 
as high as 10 mg. in twenty-four hours. It is a colorless, 
odorless liquid, very volatile, of low specific gravity. 

In testing the urine for acetone, it is usually necessary 
to distill the specimen. Occasionally, when very large quan- 
tities of acetone are present, positive reactions for acetone 
may be obtained by testing the urine directly. But in no 
case do such tests, when negative, exclude an acetonuria. 
"When tests of the urine are negative, it becomes necessary 
to distill a portion of it and to apply the tests for acetone 
to the distillate. 

Between 200 and 300 c. c. of urine are acidified with 1 
to 2 c. c. of concentrated hydrochloric acid x and distilled. 
The greater part of the acetone is contained in the first 
20 or 30 c. c. of the distillate, which is used for the tests 
to be described. If distillation of the urine is impossible. 
about 50 c. c. of urine are extracted with 20 c. c. of ether 
in a separating funnel. The urine is then allowed to es- 
cape, and to the ether about 10 c. c. of water are added. 
The fluids are well shaken. A large part of the acetone is 
in the water, which is then used for the qualitative tests. 2 

Qualitative Tests 

(1) Gunning's Test.— Five c. c. of the distillate are ren- 
dered alkaline with 5 to 10 drops of ammonium hydrate. 

1 Phosphoric acid may be used. The acid is added only to prevent the dis- 
tillation of ammonia and excessive foaming of the urine. 

2 Bohrisch, P. Pharm. ZentralhaUe, 19<j7. XLVIII. 5: 184; 206; 220; 
245. Cited by F. X. Schulz in Xeubauer-Huppert 's Analyse des Hams, 11th 
Ed., p. 252. Wiesbaden, 1910. 



THE URINE 63 

and then Lugol's solution (potassium iodid, 6 gm., iodin, 4 
gm., distilled water to 100 c. c.) or tincture of iodin is 
added until the deep black precipitate which forms no 
longer dissolves at once. This gradually disappears and is 
replaced by a yellow precipitate of iodoform crystals, rec- 
ognized by their characteristic odor and morphology. The 
crystals are often so small that the high power dry objec- 
tives of the microscope are required. They are hexagonal 
plates, often clustered in the form of six-pointed stars. 
When atypical, the crystals should be recrystallized from 
alcohol-free ether. They are colored yellow. When the 
test is applied directly to the urine the phosphates are pre- 
cipitated by the ammonia, usually in the form of crystals 
resembling fern leaves. With very small quantities of 
acetone it may be necessary to wait twenty-four hours for 
the crystals of iodoform to form. 

This test is the best qualitative test, since a positive 
reaction is obtained only with acetone. It is slightly less 
sensitive than Lieben's test. According to Bohrisch, the 
test should be applied only to the distillate, not to the urine 
directly. 

(2) Lieben's Test.— A few drops of sodium or potas- 
sium hydrate and then a little Lugol's solution are added 
to about 5 c. c. of the distillate, and the mixture is warmed. 
With large quantities of acetone, an immediate precipita- 
tion of yellow iodoform crystals (hexagonal plates or six- 
pointed stars) occurs. When the amount of acetone is 
small (0.01 mg. or less), a few hours may be required for 
the formation of the crystals, which are detected by micro- 
scopic examination of the sediment. Warming the tube in- 
tensifies the characteristic iodoform odor. The test is very 
delicate. Crystals may be demonstrable after twenty-four 
hours with as little as 0.0001 mg. of acetone. If the crys- 



64 ACETONE 

tals are atypical, the precipitate is dissolved in alcohol-free 
ether and recrystallized. 

Sources of Error. — Both alcohol and aldehyde give Lieb- 

en's test. 

(3) Legal's Test.— A few small crystals of sodium 
nitroprussid are dissolved in about 5 c. c. of the distillate. 
An excess of sodium or potassium hydrate is now added. 
If acetone is present, a red color develops, which soon 
changes to yellow. Glacial acetic acid, added in excess 
while the color is still red. causes a change to purplish red 
and finally to violet. The test indicates about 0.1 per cent, 
of acetone. 

Sources of Error. — According to v. Jaksch. paracresol 
gives a yellowish-red color with sodium nitroprussid and 
sodium hydrate : on adding an excess of glacial acetic acid 
the color changes to a rose red. and may be confused with 
the acetone reaction. Creatinin causes the same prelimi- 
nary color changes as acetone, but on acidifying with gla- 
cial acetic acid the color gradually becomes green and then 
blue. "When testing the distillate this difficulty is removed. 

(-t) Lange's Test. 1 — About 15 c. c. of urine are placed in 
a test tube and treated with 0.5 to 1 c. c. of glacial acetic 
acid. After the addition of a few drops of a freshly pre- 
pared solution of sodium nitroprussid. ammonium hydrate 
is carefully layered above the urine. In the presence of 
acetone an intense violet ring appears at the line of contact. 
The quantity of nitroprussid used is unimportant, but the 
amount added should not be enough to color the urine. The 
test, which is a modification of Legal's, is sensitive to ace- 
tone in 1 400 per cent, solution. The reaction is not given 
by alcohol or aldehyde. 

1 Lange. F. "Eine Kingprobe auf Azeton. " Miinchen. med. Wchnschr., 
1906, LITt. 1764. 



THE URINE 65 



DIAOETIC ACID 



Diacetic acid or acetoacetic acid, the precursor of ace- 
tone, is usually found in urines which contain abnormal 
amounts of acetone. When urine is allowed to stand the 
diacetic acid soon becomes converted into acetone, which in 
turn is lost within a few hours through volatilization or 
decomposition. Diacetic acid may, however, be kept in the 
urine with little loss for weeks by the addition of toluol to 
the specimen in a tightly stoppered bottle. Unless toluol 
be added, the urine should be tested for diacetic acid soon 
after it is voided. 

(1) Gerhardt's Test.— To about 20 c. c. of urine in a 
test tube add an excess of 10 per cent, ferric chlorid solu- 
tion. If a precipitate forms, it is removed by filtration. To 
the filtrate more ferric chlorid is added, as long as it pro- 
duces a perceptible darkening in color. A deep Bordeaux 
red color is produced by diacetic acid. The contents of the 
test tube are now halved, one portion being boiled, the other 
set aside as a control. If the color be due to diacetic acid 
boiling for several minutes (two or more) lessens its in- 
tensity very perceptibly, owing to the breaking up of the 
diacetic acid. 

The test indicates 0.04 to 0.05 per cent, of diacetic acid. 

Sources of Error. — After the administration of various 
drugs, notably salicylic acid, aspirin, diuretin, salol, phe- 
nacetin, acetates, formates, etc., a red color, at times indis- 
tinguishable from that produced by diacetic acid, may be 
seen. Except in the case of formates and acetates, the 
color does not fade after boiling a few minutes or standing 
several hours, as is the case when it is due to diacetic acid. 
When both diacetic acid and one of these drugs coexist, the 
urine is distilled, and the distillate tested for acetone. 



66 DIACETIC ACID 

When the disturbing body is either a formate or an acetate, 
the nrine is acidulated with sulphuric acid, cooled if neces- 
sary, and then extracted with an equal volume of ether. 
With a pipette the ether is transferred to another test tube 
and a small quantity of very dilute watery ferric chlorid 
solution added ; the tube is then well shaken. Diacetic acid 
causes a violet color in the watery layer, which changes to a 
Bordeaux reel on the addition of more ferric chlorid. The 
color fades quickly on boiling the watery layer (remove ether 
first!). Formates and acetates do not give this reaction. 
(2) Arnold's Test. 1 

Eeagents : 

Solution A. — 1 gm. of paramidoacetophenon is dissolved 
in 80 to 100 c. c. of distilled water with the aid of hydro- 
chloric acid added drop by drop during vigorous shaking. 
Acid is added till the yellow solution becomes water clear. 
An excess of hydrochloric acid is to be avoided. 

Solution B. — Sodium nitrate, 1 per cent, aqueous solu- 
tion. 

The solutions keep well. 

Method. — Two parts of solution A are mixed with 1 
part of solution B (always prepare freshly at the time of 
making the test). Add an equal volume or less of the sus- 
pected urine and then 2 to 3 drops of strong ammonia, 
shaking well. All urines give a more or less intense brown- 
ish-red color. With excessive quantity of diacetic acid the 
addition of the ammonia produces an amorphous, brown- 
ish-red precipitate, but with smaller amounts no precipi- 
tate forms. A portion of the reddish fluid is placed in a 
wine glass or test tube, and a great excess of concentrated 

1 Arnold, V. "Eine neue Reaktion zum Nachweis der Acetessigsaure im 
Ham." Wiener Min. Wclinshr., 1899, XII, 541. 



THE URINE 67 

hydrochloric acid is added (to 1 c. c. of fluid add about 10 
to 12 c. c. HC1). In the presence of diacetic acid the mix- 
ture takes on a beautiful purplish-violet color. With large 
amounts of diacetic acid the violet predominates, while 
with smaller quantities the red is more evident. With nor- 
mal urine (free of diacetic acid) only a yellow color is ob- 
tained. 

With small amounts of diacetic acid the reaction may 
fail if the urine be highly colored. In such case filter the 
urine through animal charcoal, and the reaction becomes 
positive with the water-clear filtrate. In using the filtrate 
add 2 to 3 parts of filtrate to 1 part of the mixed reagent. 

The reaction is specific for diacetic acid and its ethyl 
ester. It is not given by drugs and is more delicate than 
Gerhardt's test (Arnold). 

tf-OXYBUTYRIC ACID 

/2-oxybutyric acid, the third of the "acetone bodies," is 
found in the urine only in the presence of acetone or dia- 
cetic acid, or both, though the converse of this is not true. 
It occurs in largest amount in certain cases of diabetes mel- 
litus. Its presence may be suspected when the urine is 
found to be definitely levorotatory after fermentation of 
the glucose ; such a finding is not, of course, conclusive evi- 
dence of the presence of this body. 

(1) Black's Test. 1 — Five or 10 c. c. of urine are concen- 
trated in an evaporating dish at a gentle heat to one-third 
or one-fourth of the original volume, which eliminates the 
acetacetic acid. The residue is then acidified with a few 
drops of concentrated hydrochloric acid, and made to a 
thick paste with plaster of Paris and allowed to stand until 

1 Black, 0. F. "The detection and quantitative determination of /3-oxy- 
butyric acid in the urine. ' ' Jour. Biol. Chem., 1908, V, 207. 



68 0-OXYBUTYRIC ACID 

it begins to set. It is then stirred and broken up in the 
dish with a blunt stirring* rod. The porous meal thus ob- 
tained is extracted twice with ether by stirring and decan- 
tation. The ether extract, which contains /^-oxybutyric 
acid, is evaporated spontaneously or on the water bath. 
The residue is finally dissolved in water and neutralized 
with barium carbonate. The fluid is now poured into a test 
tube and treated with 2 or 3 drops of commercial hydrogen 
peroxid, the whole being mixed by shaking. The /3-oxj- 
butyric acid is oxidized to diacetic acid. Now add a few 
drops of 5 per cent, ferric chlorid containing a trace of 
ferrous chlorid. On standing a few seconds a beautiful 
rose color develops, which slowly intensifies until it reaches 
a maximum, and then gradually fades, owing to the further 
oxidation of the acetacetic acid. 

Sources of Error. — Black says that the chief precau- 
tions to be observed in carrying out the test are to be sure 
that the solution is cold and nearly neutral, and to avoid 
a large excess of hydrogen peroxid and iron. If too much 
of the oxidizing agents is added, and but little ^-oxybutyric 
acid is present, the color developed is transitory or fails to 
appear. By starting with a small quantity and then add- 
ing more ferric chlorid at intervals of a few minutes, until 
no further color is produced, one is able to observe the full 
intensity of color, and thereby get a rough idea as to the 
amount of ^-oxybutyric acid present. 

The test is delicate. Black found that a solution con- 
taining 0.1 mg. per cubic centimeter, or one part in 10,000, 
gave an easily recognized color. 

(2) Hart's 1 Test.— Hart adds to 20 c. c. of the suspected 
urine 20 c. c. of water and a few drops of acetic acid, and 

1 Hart, T. S. "The detection of /S-oxybutyric acid in the urine." Amer. 
Jour. Med, Sc, 1909, CXXXVII, S69. 



THE URINE 69 

boils, until the volume is reduced to about 10 c. c. To this 
residue add water to the original volume, 20 c. c. Put this 
into two test tubes (B and C) of equal size, 10 c. c. in each 
test tube. To one of the test tubes (C) add 1 c. c. of peroxid 
of hydrogen, warm gently for about one minute (do not 
boil), and then allow the fluid to cool. Add to each test 
tube 0.5 c. c. of glacial acetic acid and a few drops of a 
freshly prepared solution of sodium nitroprussid, and mix. 
Overlay the solution in each test tube with 2 c. c. of ammo- 
nium hydroxid (Lange's test, p. 64). Allow the tubes to 
stand for four or five hours, and at the end of this time 
compare them. At the point of contact between the 
ammonia and the underlying fluid, B will show no 
ring (or a faint brown ring, if much creatinin is pres- 
ent) ; test tube C, to which the hydrogen peroxid was added, 
will show a purplish-red contact ring, if /^-oxybutyric acid 
was originally present ; if ytf-oxybutyric acid was not pres- 
ent the two test tubes will not differ in appearance. If the 
two tubes are now shaken the difference in color will be 
seen throughout the fluid; this difference is intensified by 
allowing the tubes to stand for fifteen or twenty minutes 
after shaking. 

The oxidation of the 7?-oxybutyric acid to acetone by 
means of the hydrogen peroxid is said to be gradual, and 
reaches its maximum in about four or five hours, after 
which the color slowly fades. When a very large amount 
of /?-oxybutyric acid is present the difference in the two 
tubes may become evident in a few minutes. The two tubes 
should always be prepared as above. B will show whether 
all preformed acetone and diacetic acid have been driven 
off. 

The presence of sugar does not interfere with the reac- 
tion. If albumin is present, it should be removed. 



70 UROBILIN 

The method, though simpler than Black's, does not com- 
pare with the latter in delicacy. Hart finds that it will <:•-: - 
tainly detect ^-oxybutyric acid when present to the extent 
of 0.3 per cent, and probably less. 

UROBILINOGEN 

Urobilinogen is normally present in the urine in traces. 
It is converted into urobilin within a few hours after the 
urine is voided, so that it is necessary to employ fresh 
specimens in testing for it. In the twenty-four-hour speci- 
men an excess of the chromogen, though originally pres- 
ent, may be missed by the time the examination is made; 
in this case urobilin may be looked for. 

Ehrlich's Aldehyde Test. 

Eeagent : 2 

Diniethylparainidobenzaldehyde .... 2.0 gin. 

Hydrochloric acid (5 per cent.) 2 . . . .100.0 c. c. 

Dissolve. Keep in dark brown glass bottle. 

About 10 c. c. of urine in a test tube are treated with a 
few drops of the reagent. In the presence of abnormally 
large amounts of urobilinogen a red color develops in the 
cold. In normal urine the red color appears only after 
heating. If the color fails to develop on heating, urobili- 
nogen is absent. 

UROBILIN 

Urobilin, whose chromogen is urobilinogen, is a con- 
stituent of normal urine. Though it be lacking in the fresh- 
ly voided specimen, traces of it are soon present, due to 

*As the reagent does not keep well, it should be prepared in small quan- 
tity, according to the demand. 

2 About 14 c. c. cone. HC1 diluted to 100 c. c. with water. 



THE URINE 71 

the action of light on urobilinogen. Large quantities of the 
pigment may impart a deep yellowish-brown color to the 
urine, though an excess may be present without noticeable 
change in the appearance of the specimen. 

(1) Spectroscopic Determination.— When there is a 
considerable excess of urobilin it may be detected by direct 
spectroscopic examination of the urine. A small hand 
spectroscope is most convenient for the purpose. A few 
c. c. of clear urine, previously treated with a few drops of 
Lugol's solution (about 1 drop to 2 c. c), and a few drops 
of mineral acid are examined directly with the spectro- 
scope. The characteristic spectrum of acid urobilin, a 
single band (Fig. 7) between the green and the blue parts 
of the spectrum (between the lines b and F and extending a 
little to the right of F in the green), is seen. If the urine is 
very highly colored it may be necessary to dilute it before 
examining it with the spectroscope. On the other hand, 
with small amounts of urobilin the pigment should be ex- 
tracted from the acidulated urine with amyl alcohol. The 
extract then presents the band of urobilin in acid solution. 

The filtrate in the next two tests may also be examined 
spectroscopically. It must be remembered that urobilin in 
alkaline solution shows a band between b and F which is 
nearer b than that seen in acid solution. In solution with 
ammonia and zinc, the band is well seen. But if alkali be 
added to the urine for direct spectroscopic examination, 
fixed alkali will produce a darker band than ammonia. 

(2) Schlesinger's * Test.— This is the best test for rou- 
tine work. It is delicate, easily performed, and requires 
no special apparatus. 

About 10 c. c. of acid urine are treated with 5 or 6 drops 

Schlesinger, W. ' ' Zum klinischen Nachweis des Urobilins. ' ' Deutsche 
med, Wchnschr., 1903, XXIX, 561. 



72 UROBILIN 

of Lugol's solution to convert any urobilinogen present 
into urobilin. The urine is now mixed with an equal quan- 
tity of a saturated solution of zinc acetate in absolute al- 
cohol and filtered. TThen held against a dark background 
and examined with transmitted light, the filtrate shows a 
beautiful green fluorescence, whose intensity is propor- 
tional to the quantity of urobilin present. The fluid may 
be examined spectroscopically. 

The least trace of eosin or other fluorescing compound 
on the glassware may lead to misinterpretation of the re- 
sult of the test. 

The fluorescence may be made to appear more marked 
if the light be focused on the tube with a small hand lens. 

The alcoholic zinc acetate solution precipitates other 
pigments, which may interfere with the reaction. How- 
ever, when bilirubin is very abundant it is precipitated by 
adding 2 c. c. of 10 per cent, calcium chlorid solution to 
8 c. c. of urine. The mixture is filtered and the test ap- 
plied to the filtrate. The test is sensitive to 0.002 per cent, 
solutions of urobilin in urine, even in the presence of bile 
pigments. 

(3) Jaffe's Test.— The urine is treated with a few drops 
of Lugol's solution and then with an equal volume of 10 
per cent, alcoholic solution of zinc chlorid. The precipi- 
tate which forms is removed by filtration. The filtrate is 
rendered strongly alkaline with ammonia. A green flu- 
orescence denotes the rjresence of urobilin. On spectro- 
scopic examination the spectrum of urobilin in alkaline so- 
lution may also be observed. The single band is a little 
nearer b than that seen in acid solution (Fig. 7). 

The test is not as delicate as Schlesinger 's test. Zinc 
chlorid precipitates the interfering bodies less completely 
than zinc acetate, but alcoholic zinc chlorid is preferable to 



THE URINE 73 

the aqueous solution, which is frequently recommended 
(Schlesinger). 

When it is desired to determine whether urobilin is to- 
tally absent from the urine, large quantities of urine treated 
with Lugol 's solution and mineral .acid are extracted with 
a small volume of amyl alcohol, which may then be sub- 
jected to spectroscopic examination or to Schlesinger 's 
test. The aldehyde test of Ehrlich is equally decisive in 
proving the absence of the pigment if a perfectly fresh 
specimen of urine be employed. 



BILE PIGMENTS 

Bile pigments, never normally present in the urine, may 
or may not cause an appreciable alteration in its appear- 
ance, the result depending on the concentration of the col- 
oring matter. When much is present the urine has a dark 
brown, at times a greenish-brown, color. The pigments 
exist in the urine as such or as soluble combinations with 
the alkalies or alkaline phosphates. Thus it happens that 
bilirubin (hematoidin) in crystalline form is not usually 
seen in the urine; but in the urine of infants with small 
content in phosphates such crystals not infrequently form. 
They appear as yellowish or brownish-red needles or as 
rhombic plates or prisms, the latter often with rounded 
angles. The crystals are insoluble in water. In chloro- 
form, especially if hot, they dissolve readily (1:600), im- 
parting their color to the solution. In dimethylanilin bili- 
rubin crystals are very soluble (1:100). The alkaline com- 
pounds of bilirubin found in the urine are insoluble in 
chloroform. Bilirubin may, therefore, be removed from 
chloroform solution by alkali. The bilirubin as found in 



74 BILE PIGMENTS 

the urine is precipitated by the addition of hydrochloric 
acid. From ammoniacal solution bilirubin is precipitated 
by barium chlorid, lead acetate, and silver nitrate. 

Solutions of bilirubin possess no characteristic absorp- 
tion bands ; there is a continuous absorption from the red 
to the violet end of the spectrum. 

Qualitative Tests 

(1) Foam Test. — The urine is shaken vigorously and, 
if it contains considerable bile pigment, the foam presents 
a distinct yellow color. The test is practically specific, 
but is not very delicate. Similarly, icteric urines show a 
yellow staining of the sediment, and a similar color is left 
on filter paper, through which such urines have been passed. 

(2) Gmelin's Test. — The acid urine is superimposed 
carefully on yellow nitric acid 1 in a test tube ; the layering 
of the two fluids must be sharp. In the presence of bilirubin 
a play of colors is observed at the line of contact of the two 
fluids. The colors from above downward are green, blue, 
violet, red, yellow. A piece of white paper, held behind the 
test tube, with the light at the examiner's back, aids in the 
recognition of the colors. The green color is the most im- 
portant. The reaction is said to be positive in a dilution 
of 1 :80,000. 

Albumin and urobilin do not interfere with the reaction. 
Much indican may lead to confusion at times; other tests 
should then be resorted to. 

(3) Rosenbach's Modification of Gmelin's Test.— The 
urine, slightly acidified with hydrochloric acid, is passed 

1 Yellow nitric acid is quickly obtained by adding a small piece of pine or 
other soft wood to nitric acid. In a short time, nitrous acid, HN0 2 , is 
evolved. The acid should be light yellow ; too much nitrous acid may accelerate 
the oxidation to such an extent that the colors are missed entirely. 



THE URINE 75 

through a small filter paper several times. The paper is 
then unfolded and blotted lightly with dry paper. The 
stain on the paper is now touched with a drop of yellow 
nitric acid, when, in the presence of bile pigment, a play 
of colors is seen at the edge of the drop. From within 
outward the colors are yellow, red, violet, blue, and green. 
The green color, the result of oxidation of bilirubin to bili- 
verdin, is again the most important color; in its absence 
the test is negative. If the paper be allowed to dry, it 
should be moistened with a drop or two of water before 
applying the acid. The test is very delicate and practi- 
cally specific. 

(4) Huppert's Test.— In deeply pigmented urines or in 
those rich in indican or hemoglobin, this test is preferable 
to Gmelin's. The urine is made alkaline with sodium or 
ammonium carbonate, and then calcium chlorid solution is 
added as long as a precipitate forms. The mixture is 
passed through a small filter, the precipitate washed with 
water, and the precipitate and filter paper transferred to 
a test tube or porcelain evaporating dish. Acid alcohol 
(concentrated hydrochloric acid, 5 c. c, alcohol, 95 c. c.) is 
now added and carefully heated to boiling. In the pres- 
ence of bilirubin the color of the alcoholic solution changes 
to green or blue. The delicacy of the reaction varies be- 
tween a dilution of bile of 1:500,000 and 1:1,000,000 (Ham- 
marsten). 

(5) Hammarsten's Test. 

Eeagent : 

Nitric acid (25 per cent.) 1 part 

Hydrochloric acid (25 per cent.) ... 19 parts 

The reagent may be kept for about a year. It is not 
ready for use until its color becomes yellow. 



76 HEMATOPORPHYRIN 

Ordinarily it is sufficient to pour a few drops of urine 
into 2 or 3 e. c. of the reagent. Almost immediately after 
shaking, the mixture takes on a green or bluish-green color, 
which will persist about twenty-four hours. When only 
traces of bile pigment are present, 10 c. c. of the acid or 
neutral (not alkaline) urine are treated with a 10 per cent, 
solution of barium chlorid, which is added as long as a 
precipitate forms. The mixture is now centrifugalized. 
The supernatant fluid is poured off and the sediment is 
shaken with about 1 c. c. of the reagent and again centrifu- 
galized. The supernatant fluid is now a beautiful green, 
which changes upon further addition of the reagent through 
blue to violet, red, and finally reddish-yellow. The green 
color is obtained in the presence of 1 part of bile pigment 
to 500,000 to 1,000,000 parts of urine. In the presence of 
considerable amounts of blood-coloring matter or other 
pigments, a 10 per cent, solution of calcium chlorid should 
be substituted for the barium chlorid solution. 

HEMATOPORPHYRIN 

Hematoporphyrin, an iron-free derivative of hemoglo- 
bin, is a normal urinary pigment occurring in traces. When 
urine contains hematoporphyrin in considerable concentra- 
tion, its color is usually dark red. 

Garrod's Test.— One hundred c. c. of urine are treated 
with 20 c. c. of 10 per cent, sodium hydrate ; this precipi- 
tates the phosphates, which carry the pigment down with 
them. The precipitate is collected by filtration or cen- 
trifugalization, and is dissolved in acid alcohol (HC1, 5 
c. c, alcohol, 95 c. c). The solution is examined spectro- 
scopically for the bands of hematoporphyrin in acid solu- 
tion (Fig. 7), one just to the left of D, the other — a broader 



THE URINE 77 

band — between D and E. The result may be further con- 
trolled by obtaining the absorption bands of hematoporphy- 
rin in alkaline solution. The alcoholic solution is rendered 
alkaline with ammonia, acetic acid is added till the precipi- 
tate of phosphates is dissolved, and the pigment is then ex- 
tracted with chloroform. The latter is examined spectro- 
scopically for the four bands of hematoporphyrin in alka- 
line solution (Fig. 7) ; the first about midway between C 
and D, the second at D extending to the right of it, the third 
at the left of E, the fourth — a broad band — beginning at b 
and extending almost to F. 

In place of sodium hydrate, Salkowski recommends that 
the urine be treated with a solution composed of equal 
parts of cold saturated barium hydrate and 10 per cent, 
barium chlorid, while Hammarsten prefers a solution of 
barium acetate. In either case the precipitate is washed 
and then dissolved in acid alcohol, as in Garrod's test, and 
the alcoholic solution is examined for the spectrum of hem- 
atoporphyrin in acid solution. By adding an excess of 
ammonia the bands of alkaline hematoporphyrin are ob- 
tained. 

In certain instances the urine contains hematoporphyrin 
in such concentration that direct spectroscopic examination 
reveals its presence. In such case the alkaline spectrum 
is the one usually observed, though the spectrum of hem- 
atoporphyrin in acid solution may be seen, and is still 
sharper after acidifying the urine with a mineral acid. 

HEMOGLOBIN 

Hemoglobin in the urine is always pathological. The 
hemoglobin is often changed to methemoglobin. If the 
urine contains hemoglobin in relatively high concentration 

7 



78 HEMOGLOBIN 

it assumes a dark red color, which is imparted to the sedi- 
ment. The latter, however, is often dark brown. Occa- 
sionally crystals of hematoidin are found on examination 
of the sediment (see bilirubin crystals) ; when placed in a 
porcelain dish with a drop of yellow nitric acid, a play of 
colors, especially a green ring, is seen at the periphery 
of the drop (Gmelin's reaction). In hemoglobinuria red 
blood corpuscles are usually absent or present in small 
number. 

(1) Spectroscopic Determination (Fig. 7).— The urine, 
if neutral or alkaline, is rendered slightly acid with dilute 
acetic acid, and is filtered till perfectly clear. If very highly 
colored it may be necessary to dilute the specimen before 
examining it spectroscopically. 

(a) Oxyhemoglobin is characterized by the appearance 
of two bands between D and E, the narrower band being 
near D. 

(b) Reduced hemoglobin is recognized by a single broad 
band, extending from D toward E. 

(c) Methemoglobin in neutral or weakly acid solution 
produces a dark band in the spectrum between C and D, 
near C. The two additional bands seen between D and E 
are usually attributed to the coexistence of oxyhemoglobin 
in the solution. In alkaline solution methemoglobin pre- 
sents two absorption bands between D and E, resembling 
those of oxyhemoglobin, except for the fact that the nar- 
rower band in this case is situated at the right. 

(d) Hematin is found in the urine very infrequently. 
In acid solution its spectrum resembles closely that of 
methemoglobin in neutral or acid solution, consisting of a 
single dark band in the red, extending to the right of C. 
Hematin is readily differentiated from methemoglobin by 
the fact that the addition of ammonia and a reducing sub- 



THE URINE 



79 



stance, such as ammonium sulphid, to the acid solution con- 
verts the spectrum into that of hemochromogen. 



Ph O 



-'i 



B C D 



Wavelength. 7 °°|- WO 600 



Eb 




Oxyhemoglobin. 



Reduced hemoglo- 
bin. 



Methemoglobin. 



Hematin in acid 
lution. 



Reduced hematin. 



Hematoporphyrin 
in acid solution. 



Hematoporphyrin 
in alkaline solu- 
tion. 



Urobilin. 



Fig. 7. — Absorption Spectra. (After Seifert and Miiller.) 



(e) Hemochromogen [reduced hematin (d)] presents 
two dark bands, one about midway between D and E, 



80 HEMOGLOBIN 

the other to the right of E. Both bands are nearer 
the green end of the spectrum than those of oxyhemo- 
globin. 

In case the urine is very deeply pigmented, the spectro- 
scopic examination is facilitated by dilution with water, 
since concentrated solutions absorb the spectrum diffusely. 
On the other hand, with small amounts of hemoglobin, the 
delicacy of the spectroscopic test depends very largely 
upon the thickness of the layer of urine, through which the 
light passes to the spectroscope. Schumm 1 finds that with 
the usual test tube hemoglobin may be detected spectro- 
scopically in a dilution of 1 :2,000, whereas, if the urine be 
placed in the polariscope tube 10 or 20 cm. long, it is pos- 
sible to recognize one part of hemoglobin in about 25,000 
parts of urine. Eoughly, this is equivalent to one drop of 
blood in the twenty-four-hour specimen. If the oxyhemo- 
globin has been changed to methemoglobin, Schumm 
recommends the following procedure : 50 c. c. of urine, 
5 c. c. of glacial acetic acid, and 40 to 50 c. c. of ether 
are shaken together. The ether, ' after it has separated, 
is drawn off and shaken with 5 c. c. of water, which is 
then removed. The guiac test (p. 81) is then applied to 
a part of the ether extract. To the remainder add 
ammonia in excess (keep the mixture cool) and shake 
well. The ammonia layer and a part of the ether are 
allowed to run into a glass, ammonium sulphid is added, 
and the bands of alkaline hemochromogen are looked 
for. 

The following tests are applicable alike to the detection 
of hemoglobinuria and hematuria: 

1 Schumm, O. ' ' Untersuchimgen iiber den Nachweis von Blut im Harn mit 
Hilfe des spektroskopischen und einiger spektroskopisch-chemischer Ver- 
fahren. " Miinchen. med. WcMschr., 1908, LV, 1488. 



THE URINE 81 

(2) The Guiac Test. 1 

Eeagents : 

Guiac resin, powdered. 

Alcohol. 

Hydrogen peroxid or ozonized oil of turpentine. 

Tincture of guiac is freshly prepared at the time of 
making the test by adding a knife-point of powdered guiac 
to about 5 c. c. of alcohol, shaking till solution occurs. 

Equal parts of tincture of guiac and hydrogen peroxid 
are mixed and are then layered above the urine by inclin- 
ing the test tube and pouring the mixture in very slowly. 
The urine, if neutral or alkaline, is acidified with acetic 
acid before testing. An opaque ring forms at the line of 
contact between the fluids; gradually a distinct blue color 
develops. 

The test is very delicate, but it is not specific for blood. 
Disturbing substances are much less apt to be encountered 
in the urine than in the stools or gastric contents. Pus, 
when present, gives the blue color, but the reaction occurs 
without the addition of the peroxid. The urine may be 
treated with glacial acetic acid and extracted with ether 
(for details consult p. 159). For a complete list of the 
disturbing substances the reader is referred to the mono- 
graph of Kastle. 

This test is very delicate; it may be positive with 1 
part of blood in 20,000 to 40,000 parts of urine. It is chiefly 
of value when negative. A positive test does not mean the 
presence of blood — it must be confirmed by other tests ; but 
a negative reaction is conclusive evidence of the absence 

1 For a full discussion of this and allied tests, see Kastle, J. H., ' ' Chemical 
tests for blocd. ' ' Bull. No. 51, U. S. Pub. Health & Mar. Hosp. Serv., Wash., 
pp. 1-62, 1909. 



82 HEMOGLOBIN 

of blood. The activity of the guiac should be proved 
occasionally. 

Several other chromogenic substances have been used 
successfully in place of guiac, but they are all open to the 
same objection, i. e., lack of specificity. Benzidin, aloin, 
and phenolphthalin are the bodies most frequently substi- 
tuted for guiac. 

(3) Heller's Test.— The urine, if alkaline, is rendered 
neutral or slightly acid with acetic acid, and is then boiled. 
If much blood is present, a precipitate of albumin and 
hematin forms. The hot urine is now treated with so- 
dium or potassium hydrate. The phosphates are precipi- 
tated and carry down with them any hematin present. The 
latter colors the precipitate red, which constitutes a posi- 
tive reaction. In case the phosphates have already been 
precipitated from the urine, normal urine may be added to 
supply the salts in solution; or a little calcium chlorid so- 
lution is added to the urine, which is then boiled, and so- 
dium phosphate is poured into it with the sodium hydrate 
(Hammarsten). 

To prove beyond question that the red precipitate is 
caused by blood pigment, the precipitate is subjected to 
Teichmann's hemin crystal test. 

Heller's test is not very delicate, and is, therefore, less 
used now than formerly. 

(4) Teichmann's Hemin Crystal Test.— The precipitate 
obtained in Heller's test or from treating the urine with 
tannic acid is used for this test. The excess of phosphates 
may be removed by washing the precipitate with very 
dilute acetic acid. The precipitate is then dried on the fil- 
ter paper, and a small amount of it transferred to a clean 
glass slide. To it add a few small crystals of sodium chlo- 
rid. Crush the crystals and mix the powder with the pre- 



THE URINE 83 

cipitate. A cover glass is placed on the material, and gla- 
cial acetic acid is run under it. Heat the preparation just 
short of boiling % to 1 minute, replenishing the acid as 
necessary. The fluid turns brown. The specimen is al- 
lowed to cool a few minutes, and is then examined micro- 
scopically for the brown rhombic plates of hemin. It is 
often necessary to reheat the specimen several times be- 
fore the crystals are obtained. Instead of heating the speci- 
men, it may be set aside for twenty-four hours before ex- 
amining it ; in this case the crystals are usually somewhat 
larger. With small crystals, high magnification may be 
required for their recognition. 

The test is specific for hemoglobin. It often fails if too 
much sodium chlorid is added, or if the specimen is over- 
heated. 

THE DIAZO REACTION 

Ehrlich's diazo reaction is never given by normal urine, 
but is of frequent occurrence in febrile diseases, compara- 
tively rare in afebrile conditions. 

Eeagents : 

Solution 1: 

Sodium nitrite 0.5 gm. 

Distilled water 100.0 c. c. 

Dissolve. The solution does not keep well. 

Solution 2: 

Sulphanilic acid 5.0 gm. 

Hydrochloric acid, cone 50.0 c. c. 

Distilled water to 1,000.0 c. c. 

Dissolve. 



84 CHYLUPJA 

One part of solution 1 is mixed with 50 parts of solution 
2. The mixture should be prepared freshly each day, as it 
is not permanent longer than twenty-four hours. 

Equal quantities of the mixed solutions and the urine 
are mixed in a test tube. Ammonium hydrate is then run 
down the side of the tube, which is inclined, so that it forms 
a layer at the top. A red ring is formed at the line of con- 
tact of the fluids. The test tube is sealed and shaken vigor- 
ously. If the foam is colored pink, the reaction is posi- 
tive. The color fades rather rapidly. 

It is quite possible to misinterpret the result of the test, 
since a salmon or yellowish-red or brownish-red foam is 
not infrequently observed. It is essential to discard all 
results as negative unless the foam is unquestionably pink. 

At times the bodies causing the diazo reaction exist 
in the urine in such dilution that a positive reaction is not 
obtained. In such cases the test again" becomes positive, if 
the urine is concentrated to a small volume on a water 
bath. 

If the sodium nitrite solution is used in greater strength 
than 1 :50. normal urine may give the color reaction. After 
the administration of atophan | phenyl-quinolin-carboxylic 
acid), 3 gin. daily, a positive diazo test may be given by 
normal urine. 1 

CHYLURIA 

The admixture of chyle causes the urine to appear more 
or less milky, depending partly on the proportion of chyle, 
but still more on its fat content. Whether parasitic (fila- 
ria) or non-parasitic in origin, chyluria is probably always 

] Skorezewski. W., and Sohn. I. "Teber einige im Atophanharne auftre- 
tende charakteristische Eeaktionen. " Wiener Win. Wchnscnr., 1911. XXIV. 
1700. 



THE URINE 85 

due to a direct anatomical communication, between the 
lymph channels and the genitourinary tract, as Magnus- 
Levy 1 and others have pointed out. Ureteral catheteriza- 
tion often reveals the fact that the chyluria is unilateral. 
The symptom is intermittent, as a rule, depending on the 
posture of the patient ; in some cases it appears during the 
day, in others only at night. All of the normal ingredients 
of chyle may be found in the urine. They are : 

(1) Neutral Fat. — Droplets of neutral fat are always 
present; they are derived from the chyle, not from the 
blood, and the quantity found varies directly with that in- 
gested in the food. The droplets vary considerably in 
size; some are so small that they are only seen distinctly 
with the oil immersion. They possess a sharp contour 
and are highly refractive. The addition of a drop of 
Sudan III or of Scharlach E (saturated solution in 70 per 
cent, alcohol) stains the fat droplets an orange or red- 
dish-yellow color. From the alkaline urine the fat may 
be extracted by means of the usual fat solvents, such as 
ether. 

(2) Cholesterin and lecithin are found if large quan- 
tities of urine are extracted with ether. Their quantity is 
dependent largely on the food. 

(3) Sugar may or may not be discovered in the urine. 
It has been shown that chyle contains about 0.1 per cent, 
sugar in hunger or on a fat or protein diet. One part of 
chyle in two parts of urine under these circumstances 
would give about 0.03 per cent, glucose — too little to detect 
with the usual clinical tests. On the other hand, after a 
large carbohydrate meal, especially a meal containing an 
excess of sugar, the urine may contain 0.3 to 0.4 per cent. 

1 Magnus-Levy, A. "Ueber europaische Chylurie. " Ztschr. f. Iclin. Med., 
1908, LXVI, 482. 



86 FERMENTS IN THE URINE 

of glucose. With this amount glycosuria is easily recog- 
nized. 

(4) Lymphocytes are always to be seen, either in the 
sediment of the centrifugalized specimen or caught in the 
meshes of the clots, which occasionally form in the urine. 

(5) Albumin is generally found in the urine, unless the 
proportion of chyle be very small. With appropriate tests, 
such as fractional precipitation of the urine with ammo- 
nium sulphate, globulin and fibrinogen may usually be dem- 
onstrated. 

(6) Filar la Banerofti. — In parasitic chyluria the em- 
bryos of Filaria banerofti are present in the sediment or 
in the clot (see p. 117). 

LIPURIA 

Small quantities of fat are not unusual in the urine. 
When epithelial or pus cells are present it is common to 
find fat droplets in them, and a few droplets are found 
free in the urine. The term lipuria is reserved for those 
conditions in which the fat is so abundant that it is recog- 
nized macroscopically. It is important to exclude fat from 
external sources, such as dirty containers for the urine, 
the lubricant on catheters, the willful admixture of fat or 
milk for purposes of deception, etc. Fat is recognized by 
its appearance and microchemical reactions. Unless it is 
present in the form of an emulsion, it is seen on the sur- 
face of the fluid. 

FERMENTS IN THE URINE 

A number of enzymes have been discovered in the urine 
— pepsin, trypsin, lipase, diastase, etc. From a diagnostic 
standpoint diastase appears to be the most important, 



THE URINE 87 

though the determination of lipase has also been of some 
value in pancreatic disease. 

Wohlgemuth' s Method for the Determination of Dias- 
tase. 1 — A 1 per cent, starch solution is prepared. Merck's 
or Kahlbaum's soluble starch is employed. The starch 
powder is stirred in cold distilled water, which is then 
heated about 10 minutes with constant stirring, until the 
solution is clear. It is cooled and is then ready for use. 
Into each of several test tubes 5 c. c. of the starch solu- 
tion are placed with a pipette. Next add varying fractions 
of 1 c. c. of urine to the tubes, which have been numbered, 
beginning with 0.2 c. c. and decreasing gradually. Add a 
small quantity of toluol to each tube to prevent bacterial 
growth, and place the tubes in the incubator at 37° C. for 
twenty-four hours. The tubes are then removed from the 
incubator and filled almost completely with ice water. To 
each tube add 1 drop of ** iodin solution, mix well, and 
observe for the blue color of the starch-iodin reaction. The 
first tube which shows no blue is selected. From the known 
proportions of urine and starch solution in this tube, cal- 
culate the number of cubic centimeters of 1 per cent, starch 
solution which 1 c. c. of urine would convert to dextrin and 
sugar. Assuming the result to be 150, it is expressed as 
follows : D |~t° =150. This means that the urine examined 
contained sufficient diastase (D) to convert 150 c. c. of 1 
per cent, starch solution to dextrin and sugar, acting at 
37° C. for 24 hours. 

Wohlgemuth employs the urine obtained at the second 
voiding in the morning for examination. The diastase is 

1 Wohlgemuth, J. (a) "Ueber eine neue Methode zur quantitativen Be- 
stimmung des diastatischen Ferments. " ' Biochem. Ztschr., 1908, IX, 1. (b) 
' ' Untersuchungen ueber die Diastasen. Beitrag zum Verhalten der Diastase im 
Urin." Ibid., 1909, XXI, 432. (c) "Beitrag zur funktionellen Diagnostik 
des Pankreas. " Berlin. Tclin. Wchnschr., 1910, XL VII, 92. 



88 FERMENTS IN THE URINE 

greatest in the urine during fasting, and decreases three 
to four hours after meals. The highest normal value which 
he has obtained for the urine is 156. 

(2) Determination of Lipase According to Hewlett. 1 — 
Hewlett has adapted the ethyl butyrate method of Kastle 
and Loevenhart to the determination of lipase in the urine. 
The procedure follows, in the author's words: Five c. c. 
of urine are placed in each of three flasks. The urine in 
the second flask is then boiled. To the urine in the third 
flask are then added three drops of a 1 per cent, solution 
of phenolphthalein and tenth normal sodium hydrate is al- 
lowed to run in from a burette, until a faint pink color ap- 
pears throughout the fluid. The amount of sodium hydrate 
used is read off, and a like amount is added to the first and 
second flasks. To each of these two flasks, the first of un- 
boiled urine, the second of boiled urine, are then added 0.25 
c. c. of ethyl butyrate and 0.1 c. c. of toluene, and they are 
placed in a thermostat at 38° C. for about 20 hours. The 
toluene is added to prevent the growth of bacteria. At the 
end of this time each flask is taken out, and sufficient tenth 
normal hydrochloric acid is added to more than neutralize 
the alkali previously added by 0.5 c. c. Each specimen is 
then shaken in a separating funnel with 50 c. c. of redis- 
tilled ether, and the ether is separated. After adding three 
drops of a 1 per cent, solution of phenolphthalein to 25 
c. c. of pure alcohol, the latter is brought to the neutral 
point. The ether extract from the separating funnel is 
now added to the neutralized alcohol, and its acidity is de- 
termined by titrating with N-20 potassium hydrate solution 
(alcoholic). Any decided difference between the acidity of 
the ethereal extracts of the boiled and of the unboiled 

1 Hewlett, A. W. " On the occurrence of lipase in the urine as a result of 
experimental pancreatic disease." Jour. Med. Research, 1904, XI, 377. 



THE URINE 



89 



urine is due to the butyric acid formed by the cleavage of 
the ethyl butyrate; and, where the difference in acidity is 
at all great, the odor of butyric acid can be recognized. 

In normal healthy men (and dogs) the urine contains 
merely traces of lipase. The greatest difference found by 
Hewlett was 0.35 c. c. of twentieth normal potassium hy- 
drate — usually 0.1 or 0.2 c. c, 

THE URINARY SEDIMENTS 



\\ 



The sediment may be obtained for microscopic examina- 
tion by allowing it to settle in a conical specimen glass 1 
or, preferably, by centrifugalizing the urine. 
The objection to sedimentation is that cer- 
tain of the formed elements, especially casts, 
may be more or less completely digested, if 
the specimen be allowed to stand too long, 
whereas with a centrifuge the examination 
may be made almost as soon as the urine is 
passed. It is particularly in alkaline urines 
that casts rapidly disappear. It is very im- 
portant that a perfectly fresh specimen of 
urine be employed for microscopic examina- 
tion. With urine which has stood for twenty- 
four hours it is often impossible to gain a cor- 
rect impression of the sediment. Crystals, 
which were not present when the urine was Fig 
voided, may have formed, while the more im 

1 The most satisfactory sedimenting glass with which the writer is familiar 
is one designed by Dr. J. S. Brotherhood (see Fig. 8). It permits one to take 
the specific gravity without transferring the urine to another receptacle, and 
its shape insures concentration of the sediment at the bottom. The weight of 
the base is an advantage, as the glass is not easily upset. It may be obtained 
from the Arthur H. Thomas Co., Philadelphia. 




-The Syd- 
enham Sedi- 
menting Glass. 



90 THE UEINARY SEDIMENTS 

portant organized material may have become so altered 
that it is no longer recognizable. 

The sediment is removed with a pipette and several 
drops of it are transferred to a glass slide. A cover glass 
should not be placed on the specimen for the preliminary 
examination, though it may be required later. "When the 
sediment is scanty the few drops of urine adhering to the 
outer surface of the pipette should be wiped off to prevent 
dilution of the specimen. On the other hand, with abun- 
dant sediment it is often advantageous to thin it somewhat 
so that the various elements are separated and their recog- 
nition made less difficult. 

The preliminary examination of a urinary sediment * 
should always be made with low magnification and with the 
light cut off as much as possible. Usually this examination 
is sufficient. But, if all the elements in the preparation 
cannot be recognized in this way, a cover glass is placed on 
the drop of sediment, which is now examined under higher 
magnification. The oil immersion lens is not employed 
with a wet specimen, nor is it necessary. 

For microchemical tests a cover glass is placed on a 
drop of the sediment, and any excess of moisture removed 
with a blotter. With a pipette a drop of the reagent is 
placed on the slide at one side of the cover glass, while a 
piece of blotting paper is touched to the opposite side. The 
absorption of fluid by the paper creates a current, which 
draws the reagent under the cover glass. The effect upon 
the sediment is observed with the low power objective. In 
case it is necessary to use the higher power, great care 
should be exercised that the lens escapes the reagent. 

1 H. Kieder's "Atlas der klinischen Mikroscopie des Harnes" (Leipzig, 
1898) is an extremely valuable and useful reference work on urinary sedi- 
ments. 



THE URINE 91 

The Unoeganized Sediments 

For convenience the unorganized sediments are divided 
into those which occur chiefly in acid urine, and those which 
are encountered mainly when the reaction is alkaline. It 
must be remembered that the classification is by no means 
absolute; it frequently happens that a deposit, usually 
found in an acid urine, persists after the reaction has be- 
come alkaline, and, again, that a sediment which is gen- 
erally met with in alkaline urine may make its appearance 
while the reaction is still acid. 

Sediments in Acid Urine 

(1) The Quadriurates of Sodium and Potassium.— The 

quadriurates of sodium and potassium, the "amorphous 
urates/' are chiefly responsible for the pink, salmon-col- 
ored, yellow, or reddish deposits which may be seen in an 
acid urine. The salts are especially apt to be precipitated 
from concentrated specimens as they become cool. The 
precipitate absorbs the urinary pigments, urochrome (yel- 
low), and uroerythrin (red). Microscopically, the sediment 
is finely granular, the granules tending to collect in masses. 
On heating the specimen over the flame the urates go into 
solution, but are again precipitated, as the preparation 
cools. The addition of hydrochloric acid dissolves the de- 
posit ; subsequently crystals of uric acid form. The latter 
are usually colorless. The rapidity with which the uric 
acid crystals appear varies greatly ; often within ten or 
fifteen minutes they are numerous. Acetic acid also brings 
the urates into solution, but the formation of uric acid crys- 
tals may be somewhat delayed. The quadriurates give a 
positive murexid test (see p. 9). 



92 THE URINARY SEDIMENTS 

(2) Uric Acid.— Uric acid may separate from an acid 
urine from the breaking up of the quadriurates into uric 
acid and biurates. Crystals of uric acid, usually colored 
reddish or yellowish-brown, are then deposited, giving* rise 
to the so-called "brick dust" sediment. They may assume 
a great variety of form when viewed under the microscope. 
That most frequently encountered is the -'whetstone" crys- 
tal. It is seen singly or in clusters, often arranged as a 
rosette. The "church- window" shape is not uncommon. 
Ehombic plates and six-sided prisms are also characteris- 
tic. Hexagonal plates are of less frequent occurrence, and 
may be colorless; morphologically they are indistinguish- 
able from crystals of cystin. The latter, however, do not 
give the murexid test. Needles of uric acid arranged in 
sheaves are rare as a spontaneous sediment, though not 
infrequently seen after the addition of hydrochloric acid 
to the quadriurates. 

Uric acid crystals are insoluble in acetic and hydro- 
chloric acids. They are unaffected by heating the speci- 
men. The crystals are soluble in sodium or potassium hy- 
drate, and may be reprecipitated by the addition of an ex- 
cess of hydrochloric acid. They give the murexid test 
(see p. 9). 

(3) Calcium Oxalate.— Calcium oxalate crystallizes 
most frequently in acid urine, but the crystals remain after 
the reaction has become alkaline. The crystals vary con- 
siderably in size ; it is often necessary to employ high mag- 
nification to recognize them. Most often they occur as 
small, highly refractive octahedra. Depending upon the 
position of the octahedron, its form resembles a square en- 
velope or a lozenge, with lines connecting the opposite 
angles. Dumbbell or hour-glass forms, at times with ra- 
dial striations, and spheroidal masses constitute rarer 



THE URINE 93 

shapes of calcium oxalate. The crystals are usually color- 
less, but in icteric urine they may be stained yellow. Cal- 
cium oxalate crystals dissolve in hydrochloric or other min- 
eral acid, but are insoluble in acetic acid. The envelope 
forms may be mistaken for triple phosphate; the latter, 
however, are readily soluble in acetic acid. Calcium sul- 
phate, calcium carbonate, and uric acid may assume the 
hour-glass form. Microchemical tests serve to differen- 
tiate them from calcium oxalate. (1) Calcium sulphate is 
insoluble in hydrochloric acid. (2) Calcium carbonate dis- 
solves in acetic acid with the evolution of bubbles of carbon, 
dioxid, which may be seen under the cover glass. (3) Uric 
acid is insoluble in hydrochloric acid and gives the murexid 
test. 

(4) Calcium Sulphate.— Calcium sulphate (gypsum) is 
a rare deposit in very acid urine. It occurs in the form of 
long, thin, colorless needles, as long, colorless prisms, often 
arranged in clusters, or as dumbbells or hour-glass crys- 
tals. Calcium sulphate is insoluble in mineral acids and in 
ammonia. 

(5) Monocalcium Phosphate.— Monocalcium phosphate, 
acid calcium phosphate, slender, colorless, rhombic tab- 
lets, usually in clusters, resembling somewhat calcium 
sulphate, is, like the latter, of rare occurrence, and is found 
in very acid urine. The two are easily distinguished by 
the solubility of the phosphate in acetic acid and in mineral 
acids. 

(6) Hippuric Acid.— Hippuric acid crystals are also 
very rare in the urinary sediment. They are seen as color- 
less, transparent, four-sided prisms, or as needles. They 
are insoluble in hydrochloric acid, which distinguishes them 
from triple phosphate. From uric acid, which they may 
resemble somewhat, the crystals are differentiated by the 

8 



94 THE URINARY SEDIMENTS 

fact that they do not give the nrarexid test, and that they 
are soluble in alcohol and ether. 

(7) Cholesterin.— Cholesterin crystals may be found in 
the urine occasionally. They present a characteristic 
shape, being rhombic plates, often superimposed, with the 
acute angle notched, as a rule. On the addition of strong 
sulphuric acid (concentrated sulphuric acid, 5 parts, water, 
1 part), the crystals are stained carmine, which later 
changes to violet. On adding the sulphuric acid with a 
little Lugol's solution the play of colors is violet, blue, 
green, and red. The crystals are soluble in ether. 

(8) Xanthin.— Xanthin crystals have been observed in 
human urine in only a few cases. They are colorless and, 
from their shape, may be mistaken for uric acid. They 
differ from the latter in that they are soluble on heat- 
ing. They also dissolve in ammonia and give the xanthin 
test. 

Weidel's Test. — On the water bath the crystals are 
evaporated to dryness in a porcelain dish, to which chlorin 
water and a trace of nitric acid have been added. The resi- 
due, when exposed to ammonia fumes, is stained reddish 
or purplish-violet. 

(9) Hematoidin.— Hematoidin (bilirubin) crystals may 
be found in icteric urine, particularly when it is very acid. 
They are differentiated from uric acid by the reactions 
given on page 73. AYith the murexid test they give a nega- 
tive reaction. Hematoidin occurs in amorphous masses or 
as needles, often gathered together to form sheaves, or as 
rhombs, colored yellow or yellowish-brown. 

(10) Tyrosin.— Tyro sin has been found in very few 
instances as a spontaneous urinary sediment. It is found 
precipitated in the form of needles gathered together in 
bundles like sheaves of wheat. In impure state tyrosin may 



THE URINE 95 

resemble somewhat spherules of leucin. The crystals are 
soluble in alkali, in ammonia, and in mineral acids, very 
slightly soluble in acetic acid. From ammoniacal solution 
tyrosin crystallizes spontaneously on evaporation. 

To obtain tyrosin from the urine 1 the twenty-four-hour 
specimen is treated with neutral, then with basic, lead ace- 
tate, as long as a precipitate forms. The excess of lead in 
the filtrate is removed by precipitation with hydrogen sul- 
phid. The filtrate is now concentrated to small volume on 
a water bath. By fractional crystallization tyrosin and 
leucin (which usually coexist) are separated, since it is 
chiefly the tyrosin which forms the crystalline deposit. The 
crystals are then subjected to the tests for tyrosin given 
below. The leucin which is in the filtrate is converted into 
its copper salt by boiling with freshly precipitated copper 
hydroxid. From the hot solution it crystallizes in the form 
of rhombic plates. The crystals are slightly soluble in 
water, insoluble in methyl alcohol. 

Piria's Test. — Dissolve the crystals of tyrosin in warm, 
concentrated sulphuric acid, permit the solution to cool y 
then dilute with water, and, finally, neutralize with barium 
carbonate. The mixture is then filtered, and to the filtrate 
ferric chlorid solution is added. A violet color appears. 
The test may fail if free mineral acid remains or if an 
excess of ferric chlorid be added. 

Morner's Modification of Deniges' Test.— To a few c. c. 
of a reagent (consisting of 1 volume of formalin, 45 vol- 
umes of water, and 55 volumes of concentrated sulphuric 
acid) add the tyrosin crystals or solution and boil. A beau- 
tiful green color develops. 

1 This and the f olloTnng tests for tyrosin are taken from Hoppe-Seyler 's 
"Handtmch der chemischen Analyse" (H. Thierf elder), Berlin, 1909, pp. 625 
et seq., 8th edition. 



96 THE URINARY SEDIMENTS 

Hofmann's Test. — A few crystals of tyrosin are placed 
in a test tube partly filled with water, to which a few drops 
of Millon's reagent (one part of mercury dissolved in two 
parts by weight of nitric acid, sp. gr. 1.42, then warm 
gently, add two volumes of water, and, after standing sev- 
eral hours, obtain the clear supernatant fluid) have been 
added. On boiling the fluid is stained a beautiful red, and 
a red precipitate forms- 

(11) Leucin.— Leucin is not seen as a spontaneous sedi- 
ment in the urine. It is usually present with tyrosin, and 
may separate as globules resembling fat if the urine be con- 
centrated on a water bath. The globules, unlike those of 
neutral fat, are insoluble in ether. They are usually 
stained brown and present radial striations or concentric 
rings, or are hyalin. 

To obtain leucin from solution in the urine, see page 
95. For its recognition the reader is referred to works on 
biological chemistry. 

(12) Cystin.— Cystin, a sulphur-containing amino-acid, 
occurs in the urine in the form of colorless, hexagonal 
plates. The presence of the crystals constitutes cystinu- 
ria, the manifestation of a rare disturbance of intermedi- 
ary protein metabolism. (The crystals are usually found 
in neutral or alkaline urine, but may be considered here 
for the sake of convenience.) Oftentimes the crystals are 
superimposed or overlap one another. Uric acid at times 
assumes the identical crystalline form and may be color- 
less. The two may be distinguished by the fact that cystin 
is readily soluble in hydrochloric acid and ammonia; fur- 
ther, by the fact that the murexid test is not given by cys- 
tin. Cystin crystals are insoluble in acetic acid, alcohol, 
and ether. "When the crystals are atypical they may be re- 
precipitated from ammoniacal solution by the addition of 



THE URINE 97 

acetic acid. Microscopic examination should then reveal 
characteristic crystals. 

To isolate cystin in solution the urine is treated with 
neutral, then with basic, lead acetate (see under tyrosin, 
p. 95). The nitrate is concentrated on a warm bath. 
Cystin separates on prolonged standing or after the addi- 
tion of an excess of acetic acid. 

Qualitative Test. — Boil a portion of the urine with so- 
dium or potassium hydrate and lead acetate. A black color 
arises from the sulphid of lead which is formed. Albumin 
or other proteins, if present, must first be removed. 



Sediments in Neutral or Alkaline Urine 

In addition to the crystals described in acid urine, which 
frequently persist after the reaction has become alkaline, 
there are a number which are commonly found in alkaline 
urine. 

(1) Tricalcium and Trimagnesium Phosphates.— Tri- 
calcium and trimagnesium phosphate, the amorphous phos- 
phates, are recognized as white or grayish-white deposits, 
often very abundant, which are easily soluble in hydrochlo- 
ric and acetic acids. The lack of coloration and the fact 
that they do not dissolve on heating the preparation differ- 
entiate them from the quadriurates, which they resemble 
somewhat microscopically. The murexid test is negative, 
a further differential point. 

(2) Calcium Carbonate.— Calcium carbonate is also 
usually amorphous, and is generally found mixed with the 
amorphous phosphates. It differs from the phosphates in 
the fact that the addition of acid causes solution with the 
evolution of carbon dioxid. The salt may also appear as 



98 THE URINARY SEDIMENTS 

dumbbells or spheres with radiating lines, resembling sim- 
ilar forms of calcium oxalate, calcium sulphate, and uric 
acid. Its solubility in acids with gas formation identifies 
it as calcium carbonate. 

(3) Ammonio-magnesium Phosphate.— Ammonio-mag- 
nesium phosphate, "triple" phosphate, is the crystal most 
commonly observed in alkaline urine. For its formation 
it is necessary that ammonia be produced. It therefore 
happens that the crystals are occasionally encountered 
while the reaction of the urine is still acid, though their 
number rapidly increases with the progress of ammoniacal 
fermentation. The crystals belong to the rhombic system. 
The " coffin-lid " is the commonest form. Erosion of these 
produces the irregular X-shaped crystals. With good illu- 
mination triple phosphate crystals have a greenish tint. 
They vary greatly in size ; at times they are so large that 
they are visible with the unaided eye. Some of the smallest 
crystals, when perfect, resemble somewhat the envelope 
forms of calcium oxalate. Their solubility in acetic acid is 
a differential point. When the phosphates are precipitated 
artificially with ammonia, fern-like crystals are usually 
found. In a native sediment, particularly when it has stood 
for some time, it is customary to find the majority of the 
crystals imperfect. 

(4) Ammonium Biurate.— Ammonium biurate, like 
triple phosphate, is deposited only as a result of the lib- 
eration of ammonia in the urine. It forms balls or spheres 
of yellow or light brownish color, often with striations, of- 
tener with horny projections or processes, producing the 
so-called "thorn-apple" or "morning star" crystals. Their 
shape may be anything, depending on the number, posi- 
tion, and length of the projections. The crystals are so- 
luble in acetic and hydrochloric acids, with the subsequent 



THE URINE 99 

formation of uric acid crystals. They give the murexid 
test. 

It is not uncommon to find amorphous phosphates and 
carbonates, triple phosphate, and ammonium biurate com- 
bined in the sediment of an ammoniacal urine. 

(5) Neutral Magnesium Phosphate.— Neutral mag- 
nesium phosphate, dimagnesium phosphate, is a very rare 
sediment, which is met with in weakly alkaiine urine. It 
forms long, refractive, rhombic plates. On treating it with 
20 per cent, ammonium carbonate solution the crystals be- 
come opaque and the edges eroded. They are easily dis- 
solved in acetic acid, and may be reprecipitated by the ad- 
dition of sodium carbonate. 

(6) Neutral Calcium Phosphate.— Neutral calcium 
phosphate, dicalcium phosphate, is very infrequently met 
with in weakly acid, neutral, or weakly alkaline urine. It 
gives rise to colorless wedges or prisms, usually clumped 
together. The crystals are soluble in acetic acid. On 
treating them with 20 per cent, ammonium carbonate, balls 
of calcium carbonate are produced. 



The Micro chemical Reactions of Sediments to Reagents 

The reactions described above may be summarized as 
follows : 

(1) Strong acetic acid dissolves calcium and magnesium 
phosphates, ammoniomagnesium phosphate, and calcium 
carbonate, the last with the evolution of gas. It does not 
dissolve calcium sulphate, calcium oxalate, uric acid, cys- 
tin, tyrosin (very slightly soluble), and xanthin. Salts of 
uric acid are slowly eroded, and after several hours crys- 
tals of uric acid are deposited. 



100 THE URINARY SEDIMENTS 

(2) Hydrochloric acid dissolves all crystals excepting 
uric acid, hippuric acid, and calcium sulphate. 

(3) Ammonium hydrate dissolves eystin. tyrosin, and 
xanthin. Uric acid crystals are partially eroded with the 
formation of ammonium biurate. Calcium phosphate, cal- 
cium sulphate, and calcium oxalate, and the salts of uric 
acid are unaffected by ammonia. 

(4) Water in large amount dissolves calcium sulphate; 
but many other crystals are not wholly insoluble in water — 
uric acid and its salts, triple phosphate, tyrosin, and xan- 
thin. 

(5) Alcohol dissolves tyrosin, leucin, cystin. and hippu- 
ric acid. 

(6) Chloroform dissolves bilirubin (hematoidin) and 
fat. 

The Oegaxized Sediments 

(1) Epithelial Cells. — Epithelial cells are normally found 
in the urine, due to the fact that the cells of the genito- 
urinary mucosae are constantly desquamating. As a rule, 
the cells are few in number and the majority of them may 
be caught in the mucous threads of the nubecula, if the 
specimen be allowed to stand a short time. In the case of 
women, however, the urine frequently contains a macro- 
scopic sediment composed largely of enormous numbers of 
epithelial cells, derived chiefly from the vagina. 

A variety of form may be noted in the epithelial cells 
of the urine. The vaginal cells are rather large, squam- 
ous cells with relatively small, round or oval nuclei. Sheets 
of these cells are often shed en masse. Cells derived from 
the kidney are usually round or cuboidal, with large, vesicu- 
lar nucleus. 

The protoplasm of the epithelial cells is prone to under- 



THE URINE 101 

go fatty degeneration. The microscopic appearance is 
fairly characteristic. The droplets differ in size and may 
be few or numerous ; at times the cell is completely filled, 
and it may be impossible to demonstrate the nucleus. The 
fat droplets are stained a deep orange with Sudan III or 
Scharlach E. 

Occasionally myelin or albuminous granules are present 
in the protoplasm. 

Since it is quite generally agreed that it is impossible, 
from their morphology, to determine the origin of epi- 
thelial cells seen in the urine, detailed description of them 
is superfluous. 

Epithelial cells are distinguished from pus cells by the 
shape of the nucleus. The epithelial cell possesses a single 
round or oval nucleus; rarely, in disease, isolated multi- 
nucleated cells are observed. The pus cell, on the other 
hand, has a polymorphous nucleus. In the fresh sediment 
the nuclei are not easily seen ; they stand out sharply after 
the addition of dilute (3 per cent.) acetic acid. Staining 
is somewhat less satisfactory. 

" H ear t- failure" cells have recently been described in 
the urine. 1 Like those seen in the sputum, they are epi- 
thelial cells, which are laden with altered blood pigment. 
The pigment granules are light golden yellow in color. At 
times there is a diffuse yellowish staining of the cells in 
the absence of icterus. The cells are not uncommon with 
chronic passive congestion of the kidneys, but they are not 
diagnostic of the condition ; they may be found in hematu- 
rias unassociated with passive congestion. 2 The cells are 
usually more or less swollen, of varying size, often very 

1 Bittorf , A. ' ' Ueber Herzf ehlerzellen im Harne. ' ' Mimchen. med. 
Wchnschr., 1909, LIX, 1775. 

2 Roller, E. "Zum Vorkommen von l Herzf ehlerzellen ' im Harn. " Wiener 
Win. Wchnschr., 1911, XXIV, 636. 



102 THE URINARY SEDIMENTS 

large, and at times a large, round nucleus is visible (Bit- 
torf). They are frequently much degenerated. 

(2) Pus.— In health the urine may contain isolated pus 
cells, though they are ordinarily missed altogether. An ex- 
ception is not infrequently met with in women with leukor- 
rheal discharge. The vaginal secretions become mixed with 
the urine and, as numerous pus cells may be present in the 
former, they are, of course, found in the examination of 
the urinary sediment. It is, therefore, necessary in such 
cases to thoroughly cleanse the external genitals before col- 
lecting the specimen or to obtain the urine by means of a 
catheter. The second procedure is the more accurate 
method and is to be preferred. The presence in the urine 
of abnormal numbers of pus cells gives rise to the condi- 
tion designated pyuria. When only a few cells are present 
there is no macroscopic alteration in the appearance of the 
urine, but marked pyuria causes a turbidity, and in ex- 
treme cases the urine may even appear creamy. 

The pus cells (polynuclear neutrophilic leukocytes) re- 
tain their characteristic morphology well in acid urine. 
When the urine becomes strongly alkaline, the pus forms 
a ropy, tenacious mass, in which the individual cells are 
swollen, often distorted, and so greatly degenerated that 
they may be no longer recognizable. However, in weakly al- 
kaline, amphoteric, or weakly acid urines the cells are gen- 
erally very well preserved, and may even exhibit ameboid 
activity. 

Microscopically, the protoplasm of the cells is finely 
granular. The majority of the granules are the neutro- 
philic granules of the cell, though fat droplets may be more 
or less abundant. In unaltered cells the diameter is about 
12 micra — rather smaller than most of the epithelial cells. 
To determine the nature of the cells beyond question it is 



THE URINE 103 

necessary to demonstrate the typical nuclei. This is best 
accomplished by the addition of 3 per cent, acetic acid; 
the polymorphous nuclei are then sharply defined. The 
specimen is examined with the high power dry objective. 
Staining the sediment may be tried, but is less satisfac- 
tory. Carbol-thionin is one of the best stains for this pur- 
pose. 

In following a patient with pyuria, it may be desirable 
to count the pus cells in the urine from time to time. For 
this purpose the twenty-four-hour specimen should be used, 
and care must be exercised to prevent bacterial ammonia- 
cal fermentation, otherwise the cells become glued together, 
making a count impossible. The best chemical preservative 
for this purpose is formalin. Commercial 40 per cent, for- 
malin is added in sufficient quantity to give a solution of 
1 to 2 per cent., the preservative being added to each por- 
tion of urine as it is collected. Such a procedure is pos- 
sible only in a hospital, as a rule; when it cannot be car- 
ried out, 15 to 20 c. c. of formalin may be placed in the 
bottle in which the urine is collected. The formalin pre- 
vents bacterial growth and at the same time renders the 
cell nuclei more prominent. Its disadvantage lies in the 
fact that the cells are clumped together in certain instances. 
If an ice chest is available, simple refrigeration is to be 
preferred to any other method of preservation. The urine, 
if neutral or alkaline, is acidified with acetic acid. The 
specimen is well stirred to secure a uniform suspension of 
the cells, and the count is then made directly from it with 
the hemocytometer, employing the technique used for 
counting the blood. If the cells are very numerous, it 
will be found more convenient to use the red pipette. 

With the escape of a purulent exudate into the genito- 
urinary tract, the albumin of the exudate becomes mixed 



104 THE URINARY SEDIMENTS 

with the urine, constituting a false albuminuria, if the le- 
sion is extrarenal. It is often difficult to interpret findings 
when a false albuminuria, such as this, is met with. The 
question arises whether the albumin is derived entirely 
from the purulent exudate or in part from the kidneys 
(true renal albuminuria). The presence or absence of 
casts is of value in determining the latter; instrumental 
examination may be decisive. Posner x has recorded ob- 
servations which show that, with 80,000 to 100,000 pus cells 
per cubic millimeter of urine, only about 0.1 per cent, al- 
bumin is added to the urine. By comparing the cell count 
with the quantity of albumin, the source of the latter may 
be determined. 

The following chemical tests for pus may be applied to 
the urine or to the sediment: 

(a) The Guiac Test. — Equal parts of hydrogen peroxid 
and freshly prepared tincture of guiac (see p. 81), when 
layered over the urine, cause a blue ring to appear at the 
line of contact in the presence of pus. It may be necessary 
to wait a few minutes for the color to appear. The color 
disappears on boiling, unlike that caused by the presence 
of blood. The test is quite delicate, but is not specific. 

(b) Meyer's 2 Guiac Test (adapted to the urine). — A 
drop or two of the centrifugalized sediment is transferred 
to a test tube about two-thirds full of water. The contents 
of the tube are well mixed and allowed to extract a few 
minutes, in order to liberate the oxidizing enzyme of the 
pus cells. The fluid is halved. On one portion freshly pre- 
pared tincture of guiac (without hydrogen peroxid) is su- 

1 Posner, C. "Ueber Harntriibung. " Deutsche med. Wchnschr., 1897, 
XXIII, 635. 

2 Meyer, E. (a) "Beitrage zur Leukocytenf rage. " Miinchen. med. 
Wchnschr., 1903, L, 1489. (b) "Ueber die cytodiagnostische Bedeutung der 
Guajakreaktion." Ibid., 1904, LI, 1578. 



THE URINE 105 

perimposed carefully, and at the line of contact a bine 
ring appears, which fades in the course of abont a half 
hour. The remaining portion is boiled actively for two to 
three minutes, then cooled and treated with tincture of 
guiac in the manner just described. Boiling destroys the 
ferment, and the test is therefore negative. The test is 
delicate, and points definitely to the presence of an oxidase ; 
in the urine the only common source of oxidase is pus. If 
much albumin is present, the reaction may be inhibited. 1 

(3) Blood.— Red blood corpuscles are never found in 
normal, voided urine, excepting the admixtures of blood 
which occur during menstruation. Hematuria is the term 
used to signify blood in the urine. A small number of 
blood corpuscles produce no visible change in the appear- 
ance of the urine. With larger quantities the translucency 
of the urine is lost, it becomes "smoky" in appearance on 
agitating the specimen, and darker in color. A reddish- 
brown sediment composed largely of red cells may settle 
out. 

The chemical tests for blood are given in connection 
with hemoglobinuria (p. 80 et seq.). 

The microscopic examination is made with the high 
power dry objective. The erythrocytes may be well pre- 
served, and exhibit their characteristic morphology and 
color. If laking of the cells has occurred, the majority, 
or all, of the cells appear as "shadows," i. e., the color- 
ing matter has escaped from the red cell, and only the cell 
membrane remains. To detect the shadows it is essential 
that the light be cut off as much as possible. In concen- 
trated urine crenation of the red cells, giving rise to thorn- 
apple forms, is observed. 

(4) Casts.— The occurrence of casts in the urine, cylin- 

1 Watson, Helen. Personal communication. 



106 THE URINARY SEDIMENTS 

druria, 1 is very frequent in disease, and may also be ob- 
served in old age and in association with so-called physio- 
logical albuminurias. The casts are derived from the renal 
tubules. They vary greatly in size, the longest measuring 
in the neighborhood of 1 mm. The thickness of casts is 
also variable, but in a given cast the width is quite uniform. 

(a) Epithelial casts are composed of renal epithelial 
cells in whole or in part. Any cast to which one or more 
renal epithelial cells are attached may conveniently be des- 
ignated epithelial (Emerson). The cells are not of equal 
size, some being large, others smaller. They have a round 
or oval nucleus, and are usually flat and polygonal. In 
most instances the protoplasm of the cells is degenerated, 
showing fat droplets, albumin granules, or, more rarely, 
myelin droplets. It is unusual to find true epithelial cylin- 
ders possessing a distinct lumen. To distinguish between 
epithelial and pus cells it is necessary to demonstrate the 
morphology of the cell nuclei by the addition of dilute 
acetic acid. The cast may be mixed, i. e., it may contain 
both epithelial and pus cells, it may be partly cellular, 
partly granular, etc. 

(b) Pus casts, like epithelial casts, consist in whole or 
in part of pus cells. The cells are generally smaller and 
rounder than the epithelial cells. Their protoplasm is fine- 
ly granular, but is subject to the same degenerations as 
that of epithelial cells. The cells are characterized by their 
polymorphous nuclei, which are usually visible only after 
treating the specimen with dilute acetic acid or a dye. At 
times the cells are so degenerated that the nuclei are no 
longer demonstrable. 

(c) Blood casts, when pure, are clots which form in the 
renal tubules. However, any cast in which one or more 

Emerson, C. P. ' ' Cylindroma. " Jour. A. M. A., 1906, XLVI, 5; 89. 



THE URINE 107 

blood cells are visible is designated a blood cast. At times 
the red blood corpuscles are not well preserved; shadows 
of red cells and grannies or crystals of hematoidin may 
be attached to the cast. 

(d) Fatty casts resnlt from the fatty degeneration of 
the cells of epithelial casts, or, less commonly, of pns casts. 
Often the outlines of the original cells are preserved. The 
casts usually have a yellowish or even brownish tint. The 
droplets vary considerably in size, some being almost as 
large as a cell. Ether dissolves the fat droplets, and they 
may be stained by adding Sudan III or Scharlach E to the 
preparation. Occasionally fatty acid needles project from 
the cast. 

(e) Coarsely granular casts are whitish, yellow, or very 
dark brown in color, quite opaque, and are covered, either 
partly or entirely, by coarse granules, as. their name indi- 
cates. Some of the granules dissolve in ether and stain 
with osmic acid or other fat stains, while others are al- 
buminous and are soluble in acetic acid. Occasionally 
granules resembling myelin droplets are observed. Coarse- 
ly granular casts are probably derived from epithelial and 
pus cell casts. All stages in transition may be seen. The 
casts are often partly waxy. 

(f ) Finely granular casts resemble the coarsely granu- 
lar, but they are much less opaque and the granules are 
much finer. Transitions from the coarsely to the finely 
granular are met with. The granules may cover part or 
all of the cast. The non-granular portion of the cast may 
be cellular; more frequently, it is hyalin. Fat droplets 
are of much less frequent occurrence than in the coarsely 
granular variety, and myelin droplets are exceptional. The 
finely granular casts are best seen with low illumination. 
They are one of the commonest types of cast in disease. 



108 THE URINARY SEDIMENTS 

(g) Hemoglobin casts are very rare and are always as- 
sociated with hemoglobinuria. They are covered with dark, 
granular pigment; less commonly needles of hematoidin 
are attached to the casts. 

(h) Waxy (colloid or amyloid) casts are opaque, very 
refractive, and white or yellowish in color. Their appear- 
ance suggests bodies made of paraffin or wax, according to 
the color of the cast. They are very brittle, and not un- 
commonly present transverse fissures or cracks. During 
centrifugalization they may be broken; the fragments are 
then seen in the sediment. Some waxy casts give the iodin 
reaction for amyloid when treated with LugoPs solution. 
It is not unusual to find the cast in the form of a spiral or 
corkscrew. Cells may be attached to waxy casts, or they 
may be granular in part. They are supposed to be de- 
rived from coarsely granular casts or from hyalin casts 
which have remained in the renal tubules for some time. 

(i) Hyalin casts are pale and very slightly refractive. 
Unless most of the light is cut off, it is impossible to see 
them; they are almost glassy in their translucency. They 
may be stained by dilute gentian violet or by Lugol's solu- 
tion, and are then easily found. They are usually narrow 
and have rounded ends. In a urine undergoing ammoniacal 
fermentation they disappear more rapidly than any other 
kind of cast. Between the glassy hyalin cast and the waxy 
cast there is a large intermediate group, consisting of casts 
which are much less opaque than waxy casts, but — though 
designated hyalin — possessing considerably more density 
than the glassy t}^pe of hyalin cast. Hyalin casts are sup- 
posed to represent albumin coagula from the renal tubules 
or a morbid, coagulable secretion of the renal cells. They 
are the commonest variety of cast. 

Upon any cast granular, amorphous urates may be de- 



THE URINE 109 

posited, producing a finely granular appearance. Bacteria 
in large number, attached to a cast, produce a somewhat 
similar picture. The uniform outline of the cast is often 
lost, and the artefact may be recognized as well by micro- 
chemical reactions. 

(]) Cylindroids are casts, one of whose ends tapers to a 
thread-like filament. They are usually hyalin, though at 
times granular, and have the same significance as casts. 

Under the name pseudocast is included anything which 
may be mistaken for a cast. Scratches on the glass slide 
most often mislead the beginner. Particles of dust, fibers, 
etc., may also cause confusion. The morphology of the cast 
is not duplicated, and experience soon teaches one to differ- 
entiate. 

(5) Mucous Threads.— Mucous threads, sometimes in- 
cluded with cylindroids, with which they have nothing in 
common, are normally present in the urine. They make 
up the nubecula. They appear as long, narrow, translucent 
bands of mucin, of unequal thickness, often twisted or 
folded like a ribbon, at times branched. Nearly always a 
few epithelial cells and, perhaps, an occasional pus cell 
are attached to the threads. Unlike hyalin casts and cylin- 
droids, mucous threads are insoluble in acetic acid. The 
length of the threads is at times great; a single specimen 
may extend through several fields of the microscope. 

(6) "Clap Threads." — Clap threads {Tripp erf dden) 
are white or grayish-white, thread-like bodies which are 
seen floating in the urine, when the specimen is agitated. 
They are not always gonorrheal in origin, as the name sug- 
gests, though in the vast majority of instances associated 
with a chronic specific urethritis. They are y 2 to 1 cm. or 
more long, and consist of a matrix of mucus, in which epi- 
thelial or pus cells or both are embedded. The pus cells 



110 MICROORGANISMS IX THE URINE 

may be so abundant that the thread is quite opaque and 
yellowish. In the gonorrheal eases it is often possible to 
demonstrate the presence of the gonococcus. 

MICROORGANISMS IN THE URINE 

Gonococcus.— The gonococcus is a diplococcus shaped 
like a biscuit or coffee bean. It is found either within the 
pus cell- or free in the serum. It is Gram-negative. There- 
fore, the finding of a diplococcus in urethral pus, of charac- 
teristic morphology, which decolorizes with Gram's stain, 
makes it highly probable that the organism is the gono- 
coccus. 

To demonstrate the gonococcus the following procedure 
may be employed : 

(1) A smear of the pus is made on a glass slide. It is 
dried in the air and fixed by passing it through the flame 
of a Bunsen burner five or six times. 

(2) Stain 1 to 3 seconds with anilin water gentian vio- 
let. (Avoid overstaining.) (To prepare anilin water gen- 
tian violet, 10 parts of anilin oil are thoroughly shaken 
with 100 parts of water, and. after standing about five 
minutes, the rather milky emulsion is filtered through a 
moistened filter paper. The filtrate should contain no large 
oil droplets. Now add 11 parts of saturated alcoholic solu- 
tion of gentian violet and 10 parts of absolute alcohol. The 
solution keeps not longer than eight to ten days. — Schmorl.) 

(3) "Wash the preparation immediately in tap water and 
blot it till dry. 

(4) Add a drop of immersion oil, and examine the speci- 
men microscopically for diplococci. If none is found after 
examining three slides carefully, the chances are that diplo- 
cocci are not the etiological factor in the production of the 
purulent exudate. If suspicious organisms are seen, it be- 



THE UEINE 111 

comes necessary to determine whether they are Gram-nega- 
tive, i. e., whether they are decolorized after treating them 
with Gram's iodin solution. The further steps are: 

(5) Eemoval of the oil by wiping the specimen with 
xylol. 

(6) The specimen is now covered with Gram's iodin 
solution, one to two minutes (iodin, 1.0 gm. ; potassium 
iodid, 2.0 gm. ; distilled water, 300.0 c. c), and then, with- 
out washing in water, it is transferred to — 

(7) Absolute alcohol, in which it is decolorized, until the 
specimen is colorless or yellowish-gray, excepting the 
thickest parts of the smear, which may still retain a little 
blue. Blot dry. 

(8) Counterstaining may be performed with Bismarck 
brown (vesuvin). [The stain is prepared by dissolving 2 
gm. of the powdered stain in a mixture composed of 60 
c. c. of 96 per cent, alcohol and 40 c. c. of distilled water. 
The solution is boiled carefully, and, after it has cooled, is 
filtered. To prevent bacterial growth, a few drops of car- 
bolic acid may be added (Schmorl).] The stain is allowed 
to act one to two minutes. 

(9) Wash in water, dry, and examine. 

After the first staining all bacteria and cell nuclei are 
colored purple by the gentian violet. Decolorization re- 
moves the dye from all Gram-negative bacteria and from 
the cell nuclei. The counterstaining with Bismarck brown 
then stains the Gram-negative bacteria and nuclei brown, 
whereas the Gram-positive organisms retain the violet. 

Treponema Pallidum (Fig. 9).— Treponema pallidum 
{Spirochceta pallida), the specific parasite of syphilis, 
may be sought in the serum obtained from specific lesions. 
The surface of the lesion is first cleansed to remove Spiro- 
chseta refringens and other contaminations as much as pos- 



112 



MICROORGANISMS IN THE URINE 



sible, and then, if necessary, the lesion is slightly scarified 
or rubbed with sterile gauze. By pressure or, better still, 
by suction apparatus a drop of serum usually slightly blood- 
stained is obtained. Many red corpuscles render the exam- 
ination difficult and are to be avoided. The serum may be 
examined in the fresh state with a dark field illuminator, or 
preparations may be stained. 




Fig. 9. — Treponema pallidum (Spirochseta pallida) on the left; Spiroch^eta re- 
fringens on the right. (After Emerson.) 



The Treponema pallidum has a length of 4 to 10 to 20 
micra, is very delicate (0.5 micron or less in thickness), 
has a spiral form, the turns being numerous and close to- 
gether, and, in the fresh specimen, has a screw-like motion. 
In spite of its motility its position in the field remains al- 
most stationary. The organism stains very faintly, as its 
specific name indicates, and is difficult to see. 

Staining Methods. — Smears of the serum are prepared 
on glass slides or cover glasses, and allowed to dry in the air. 

(1) The smears may be fixed and stained with many of 
the modifications of the Eomanowsky stain, such as 



THE URINE 113 

Wright's, Leishman's, Wilson's, Hasting 's. 1 The tech- 
nique is the same as that used in staining the blood (p. 
285). The Treponema pallidum is usually stained a faint 
blue, but occasionally has a pinkish color, while Spirochseta 
refringens is stained a deep blue. 

(2) Giemsa's stain is also a modification of the Eoman- 
owsky stain. It has been employed extensively in search- 
ing for the spirochetes. Of the numerous methods of using 
it, the following are recommended: 

(a) The specimens 2 are fixed by immersion in absolute 
alcohol 15 to 20 minutes or by passing through the flame 
three times. Ten drops of Giemsa's stain (Griibler's mix- 
ture) are then mixed with 10 c. c. of distilled water, shak- 
ing after the addition of each drop of stain. (The dilution 
must be freshly prepared each time the stain is used.) 
Cover the specimen with the diluted stain, warm it till a 
slight steam arises, allow it to cool about 15 seconds ; the 
stain is then poured off, and replaced by more of the diluted 
stain. This procedure is repeated four or five times, when 
the specimen is washed, dried, and mounted in balsam. 
The parasites are stained dark red. The slide must be free 
from grease, and the receptacle for the diluted stain and 
the staining forceps must be free from acid or precipitated 
stain. The water used for washing must not be acid. 

(b) Giemsa's 3 azure-eosin staining mixture (Griibler's 
make) is diluted with an equal volume of pure methyl al- 
cohol (Kahlbaum's or Merck's) and placed in a dropping 

1 Geraghty, J. T. ' ' The practical value of the demonstration of Spiro- 
chasta pallida in the early diagnosis of syphilis." Bull. Johns Hopkins Hosp., 
1908, XIX, 364. 

2 From Mallory, F. B., and Wright, J. H. "Pathological Technique." 
Philadelphia and London, 5th edition, 1911, p. 418. 

3 Giemsa, G. ' ' Ueber eine neue Schnellf arbung mit meiner Azur-eosin- 
losung." Munchen. med. Wchnschr., 1910, LVII, 2476. 



114 MICROORGANISMS IN THE URINE 

bottle. It is well to prepare only a small quantity at a time, 
as it is not known how permanent the solution is. The air- 
dried films are then placed in a small Petri dish with the 
specimen side up. The film is now covered with 10 to 15 
drops of the alcoholic staining mixture for y 2 minute. The 
preparation is thus fixed and the staining is begun. Add 
enough distilled water to cover the specimen (usually 10 
to 15 c. c), and agitate the dish till a homogeneous mixture 
of the stain is secured. Allow the specimen to remain in 
this mixture 5 minutes. The film is now washed in dis- 
tilled water, dried, and mounted in balsam. The spiro- 
chetes are stained pink. 

(3) Stern's 1 Silver Impregnation Method. — The smears 
of serum, air-dried, are first placed in the incubator at 
37° C. for a few hours. They are then transferred to a 
colorless glass filled with 10 per cent, aqueous solution of 
silver nitrate, and exposed to diffuse daylight for several 
hours. The preparation gradually assumes a brown color. 
When this has reached a certain shade (quickly learned by 
practice) and the film shows a metallic luster, it is removed 
from the silver and washed in distilled water. In a prop- 
erly treated specimen the spirochetes are stained deep 
black on a pale brown or colorless background. The organ- 
isms are slightly thicker than in specimens stained with 
Giemsa 's stain. Anomalies in staining may be encountered. 
At times the spirals are more deeply stained at the upper 
bend of the curve than at the lower, which then appears 
gray. Or there may be only a row of deep black granules 
or dots representing a spirochete. The erythrocytes are 
well preserved, show a delicate, black contour, and present 
a number of fine granules. , 

1 Stern, M. ' ' Ueber den Xachweis der Spiroehaeta pallida im Austrich 
mittelst der Silbermethode. ' ' Berlin. Uin. Wclinschr., 1907, XLIV, 400. 



THE URINE 115 

The specimen should not be exposed to direct sunlight 
while it remains in the silver, for, though the preparation 
quickly becomes dark and even black, the spirochetes are 
unstained. 

(4) Burri's India Ink Method. 1 — One loopful of serum 
is mixed on a glass slide with a loopful of India ink, and 
spread in a thin film by means of a second slide. The slides 
must be perfectly clean. The film, which is allowed to dry 
in the air, should be dark brown or black. A drop of im- 
mersion oil is placed on the specimen, which is ready for 
examination. Bacteria, spirochetes, blood corpuscles, etc., 
are unstained, and appear as refractive bodies on the dark 
background. According to Colin and others, the best re- 
sults are obtained with Gun ther- Wagner's Chin-Chin black 
pearl ink, though fair success may be had with other inks, 
as Carter's or Higgin's. 

Bacillus Tuberculosis.— The Bacillus tuberculosis is 
not easily recognized in the urine because of the constant 
presence of the smegma bacillus on the genitalia, an organ- 
ism whose morphology is quite similar to that of the tu- 
bercle bacillus, and which cannot be separated from the lat- 
ter with certainty by staining. It is, therefore, necessary 
to exclude the smegma bacillus from the urine as a prelim- 
inary step in the examination for the tubercle bacillus, as 
Young and Churchman 2 have shown. The technique which 
these authors have developed and which has proved reli- 
able is as follows : The foreskin, if present, is rolled back 
and the glans penis is washed thoroughly with green soap 

1 Cohn, J. S. i ' On the means of finding the Spirochseta pallida with spe- 
cial reference to the India ink method. " Interstate Med. Jour., 1911, XVIII, 
26. 

2 Young, H. H., and Churchman, J. W. ' ' The possibility of avoiding con- 
fusion by the smegma bacillus in the diagnosis of urinary and genital tubercu- 
losis. " Amer. Jour. Med. Sci., 1905, CXXX, 52. 



116 ANIMAL PARASITES 

and water, using large amounts of water for the rinsing. 
The irrigating catheter is now introduced about six inches 
into the urethra (to the triangular ligament), while the 
patient keeps the sphincter urethral closed to prevent fluid 
entering the bladder. About one quart of sterile water is 
employed in the irrigation of the urethra. Since the smeg- 
ma bacillus is not found back of the triangular ligament, 
the urinary tract is practically freed of this organism by 
the procedure just described. The patient is now in- 
structed to urinate into three glasses, and a portion of the 
urine from the third glass is centrifugalized at least five 
minutes at high speed. Three smears of the sediment 
obtained are made and stained for tubercle bacilli by the 
Ziehl-Xeelsen method (p. 213). If a thorough examina- 
tion of the stained specimens reveals no acid-fast bacilli, 
the result of this particular examination is reported as 
negative. 

Occasionally pus is so abundant that the search for tu- 
bercle bacilli is very difficult. When such is the case the 
antiformin method may be resorted to (p. 214). The pus 
cells are completely dissolved. It must be remembered that 
all acid-fast bacteria are resistant to the action of anti- 
formin, so that it is necessary to observe the usual precau- 
tions to exclude the smegma bacillus. 



ANIMAL PARASITES IN THE URINARY PASSAGES 

(1) Trichomonas Vaginalis.— Trichomonas vaginalis, 1 
a flagellate closely allied to Trichomonas intestinalis, 
though probably not identical with it, may be found 

1 Dock, G. ' ' Trichomonas as a parasite of man. ' ' Amer. Jour. Med. Sci., 
1896, CXXXI, 1. 



THE URINE 117 

in the vagina and occasionally in the bladder. In either 
locality it comes in contact with the nrine, in which it may 
be found. It thrives only in an acid medium; this is sup- 
plied by the normal vagina, except during the menstrual 
period, when the mucosa is bathed in the bloody discharge. 
Trichomonas vaginalis is a pear-shaped organism with 
pointed extremity and, at the anterior rounded end, pre- 
sents four flagella. An undulating membrane is also pres- 
ent. It measures usually 0.015 to 0.022 mm. in length and 
0.010 to 0.015 mm. in width, though larger and smaller 

forms occur. It appears to be a 
specific parasite of the female sex. 
(2) Filaria Bancrofti.— Filaria 
bancrofti (Fig. 10) is of common 
occurrence in tropical and sub- 
tropical countries. Its embryos 
may be found in the urine in cases 
of parasitic chyluria. They are 

Fig. 10.-Embryo of Filaria eitller free in tlie Urine and aC " 

bancrofti. x 50. (After tively motile in a fresh specimen, 

Emerson.) J r 

or caught in the clot. The em- 
bryos are 0.125 to 0.3 mm. long, with a thickness of 0.007 
to 0.011 mm. (Blanchard). When found or suspected in the 
urine, the diagnosis should be confirmed by examination of 
the patient's blood (p. 310). 

(3) Dioctophyme Renale.— Dioctophyme renale (Eu- 
strongylus gigas), another nematode, is excessively rare 
in man, though not very uncommon in dogs in this country. 
It is the largest round-worm parasitic in man. Its habitat 
is the kidney. Lodging in the pelvis of the kidney, it pro- 
duces a pressure atrophy until, when the parasite reaches 
maturity, little or none of the parenchyma of the kidney re- 
mains. The male measures 14 to 35 cm. in length, with a 




118 ANIMAL PARASITES 

thickness of 0.4 to 0.6 era. The female is much larger — 
25 to 100 cm. long and 0.4 to 1.2 cm. thick, and is bright 
red in color. Infection is diagnosed by finding the ova 
(Fig. 11) in the urine. The latter are oval, 0.064 to 0.068 
mm. in their long axis by 0.042 to 0.044 mm. in the short 
(Blanchard). The shell is covered with an albuminous 
coating, which is stained brown and is thrown into ridges, 
making the surface of the egg appear more uneven than 
that of Ascaris lumbricoides, which it resembles somewhat. 




Fig. 11. — Ova of Dioctophyme rexale. X-400. (After Emerson.) 

The albuminous coating is lacking at the poles of the ovum 
and the latter appear colorless. 

(4) Schistosoma Hematobium.— Schistosoma hemato- 
bium (Bilharzia hamiatobia), 1 a trematode, is an important 
urinary parasite in tropical and subtropical climates. It is 
especially prevalent in Egypt. The parasite lives in the 
veins of the urinary bladder ; it deposits its ova in the 
veins. The ova then pass from the veins to the bladder. 
The ova are similar to those found in the feces (q. v.), ex- 
cept for the fact that the spine is terminal instead of sub- 
terminal (Fig. 26). As the sharp-sinned ova pierce the 
wall of the vein, hemorrhage, of course, ensues, with the 
result that hematuria is a quite constant symptom of the 
infection. 

x Lane, C. G. " Bilharziasis ; report of a case with appendicitis; literature 
since 1904." Boston Med. and Surg. Jour., 1911, CLXIII, 937. 



THE URINE 119 

PROSTATIC FLUID 

Prostatic fluid 1 is obtained by massage of the prostate 
gland per rectum, the urethra having been irrigated pre- 
viously. The amount of fluid obtained at a " milking " 
varies from a few drops to 4 or 5 c. c. The fluid is of low 
specific gravity, slightly tenacious, grayish-white, yellow- 
ish, or greenish in color, and usually has a milky turbidity 
from the lecithin granules contained in it. 

A fresh drop of the fluid is examined microscopically 
for the presence of motile spermatozoa. Lecithin granules 
vary considerably in size. The smallest are minute specks, 
the largest four micra or more in diameter. They are 
moderately refractive. Corpora amylacea, laminated bod- 
ies with a granular center, may be met with, especially in 
specimens obtained from the aged. They resemble starch 
granules not only in form, but also in the fact that they 
may be stained blue with iodin. Various kinds of epi- 
thelial cells may be found. In examining for epithelial 
and pus cells it is well to add dilute acetic acid to bring 
out the cell nuclei. Spermin crystals (Bottcher's crystals), 
transparent needles or whet-stones, are observed at times. 
They may resemble Charcot-Leyden crystals, but differ 
from the latter in that they are soluble in alkalies and in 
formaldehyde. 

FUNCTIONAL DIAGNOSIS OF THE KIDNEY 

Many tests, some simple, others complicated, have been 
introduced to measure the functional capacity of the kid- 
neys. All have had certain well-recognized limitations, and 

1 From Emerson, C. P. ' ' Clinical Diagnosis. ' ' 



120 FUNCTIONAL KIDNEY DIAGNOSIS 

none has been particularly helpful where the two kidneys 
are equally involved in the disease process, as in nephri- 
tis, until Bowntree and Geraghty described their phenol- 
sulphonephthalein test. This constitutes by far the most 
satisfactory and exact method of functional diagnosis, and, 
in the hands of a number of workers, has proved of im- 
mense value in the diagnosis, prognosis, and treatment of 
both medical and surgical diseases of the kidneys. In sur- 
gical affections some of the simpler tests may be used in 
conjunction with the "phthalein" test. The specimens ob- 
tained by ureteral catheterization often permit of accurate 
diagnostic conclusions through comparison of the micro- 
scopic and chemical findings from each kidney. Urea de- 
terminations with the hypobromite method are frequently 
made to advantage ; for, though the values obtained repre- 
sent total nitrogen more nearly than urea, nevertheless the 
comparative efficiency of the two kidneys may be fairly 
accurately determined in many instances. The information 
thus gained is practically always corroborated by the 
phthalein test, but frequently the latter will give evidence 
of disease when other tests are misleading. In nephritis 
and analogous conditions, where each kidney is involved to 
about the same extent, the phthalein test is the only reli- 
able measure of functional capacity. 

The Phthalein Test of Rowntree and Geraghty. 1 — 
Twenty to 30 minutes before starting the test the patient 
is given 300 to 400 c. c. of water to insure free urinary se- 
cretion. Then the bladder is catheterized with aseptic tech- 
nique, and 1 c. c. of a solution containing 6 mg. of phenol- 

1 Eowntree, L. G., and Geraghty, J. T. (a) "An experimental and clinical 
study of the functional activity of the kidneys by means of phenolsulphone- 
phthalein. " Jour. Plmrm. 4' Exp. Thera-p., 1910, I, 579. (b) "The sulphone- 
phthalein test for estimating renal function. ' ' Jour. A. M. A., 1911, LVII, 811. 
(c) "The phthalein test."' Arch. Int. Med., 1912, IX, 281. 



THE URINE 121 

sulphonephthalein 1 is administered intramuscularly into 
the lumbar muscles. (The solution is prepared as follows: 
"0.6 gm. of phenolsulphonephthalein and 0.84 c. c. of £ 
sodium hydrate are diluted with 0.75 per cent, sodium chlo- 
rid solution up to 100 c. c. This gives the monosodium or 
acid salt, which is red in color, and which is slightly irri- 
tant locally when injected. It is necessary, therefore, to 
add 0.15 c. c. more of the £ hydroxid, a quantity sufficient 
to change the color to a beautiful Bordeaux red. This 
preparation is non-irritant.") 

The catheter is retained until the dye appears in the 
urine, when it may be withdrawn if there be no obstruction, 
as from enlargement of the prostate. The urine is col- 
lected in a vessel, which contains one drop of 25 per cent, 
sodium hydrate, since the red color of the drug is appar- 
ent only when the reaction of the solution is alkaline. 
The time of appearance of the dye in the urine is noted. 
At the end of the first hour after administering the phthal- 
ein the patient urinates into a clean receptacle, and into a 
second receptacle at the end of the second hour. Each 
specimen is now rendered distinctly alkaline by the addi- 
tion of 25 per cent, sodium hydrate in order to elicit the 
maximal color. The dye is yellow or orange in an acid 
urine, but becomes purplish-red when the reaction is al- 
kaline. Place the urine (each specimen separately) in a 
volumetric flask of 1,000 c. c. capacity, and add distilled 
water to 1,000 c. c. Mix thoroughly, and filter a small por- 
tion for comparison with the standard solution. When the 
ureters are catheterized four specimens are obtained in the 
two hours. Each is examined colorimetrically for phthal- 

1 The substance is supplied by Hynson, Westcott & Co., Charles and 
Franklin Sts., Baltimore, Md. It is dispensed in glass ampuls. The dose is 
1 c. c. 



122 FUNCTIONAL KIDNEY DIAGNOSIS 

ein content. The functional capacity of each kidney is de- 
termined, and the sum of the four determinations indicates 
the total function. 

The standard solution is an aqueous solution of phenol- 
sulphonephthaiein containing 6 mg. to the liter, as de- 
scribed above, the solution being rendered strongly alka- 
line. Colorimetric determination of the quantity of drug 
excreted in a given specimen is made with the Dubo£ 
colorimeter or with Eowntree and Geraghty *s modification 
of the Autenrieth-Konigsberger colorimeter. 1 Employ- 
ing the Autenrieth-Konigsberger instrument (Fig. 12), the 
standard solution is placed in the wedge-shaped glass. A 
filtered portion of the urine, rendered alkaline and diluted 
to one liter, as described, is poured into the rectangular 
glass. In one side of the case of the instrument there is 
a narrow slit, the opposite side being frosted glass. With 
the frosted glass held to the light, the observer looks 
through the slit and sees the two columns of fluid — urine 
and standard solution. By means of a thumb-screw, the 
wedge containing the standard solution is elevated or low- 
ered until the color intensity is alike on the two sides. The 
percentage of coloring matter in the urine is now read di- 
rectly from the position of the indicator on the scale. 

As a routine procedure Eowntree and Geraghty recom- 
mend intramuscular (lumbar) injection of the drug. They 
employ a Eecord syringe of 2 c. c. capacity graduated in 
4 c. c. ''Whereas in the case of phthalein a normal kid- 
ney excretes the greater part of the dye injected within 
two hours of the time of its administration, and then only 
a small trace for the next two hours, the moderately dis- 

1 This instrument costs about one-fifth as much as the Duboscq colorimeter 
and is perfectly satisfactory. The Eowntree and Geraghty modification may be 
had from Hynson. Westcott & Co., Baltimore. Md. It is made by Hellige in 
Freiburg. 



THE URINE 



123 



eased kidney secretes a fair amount within the first two 
hours, say 50 per cent, of that excreted by the normal kid- 
ney, but, the concentration in the blood still being high, 
it continues to excrete a fair amount in the following two 




Fig. 12. — The Autenrieth-Konigsberger Colorimeter as Modified by Rown- 
tree and geraghty for the determination of phenolsulphonephthalein. 

hours, so that at the end of four hours little difference may 
exist in the total work accomplished. One-hour and, at the 
most, two-hour observations are, therefore, recommended. 
In cases in which only slight changes in function exist this 
can be most accurately demonstrated by one-hour collection 
following the use of an intramuscular (lumbar) injection." 
With intravenous injection the time of appearance and the 
duration of maximal elimination are shortened, but the re- 
sults are, on the whole, less trustworthy. 



124 FUNCTIONAL KIDNEY DIAGNOSIS 

With normal kidneys the following findings have been 
obtained : 

Time of 
Ad stration Appearance Quantity Excreted 

Intramuscular (lumbar) 5-11 min. 51.8-64.1% first hour 

60-85% two hours 
Intravenous 3-5 " 3445% in 1st 15 min. 

50-65% in 1st 30 min. 
63-80% in 1st 60 min. 

As the intensity of color of the dye gradually dimin- 
ishes in alkaline urine, it is necessary that the determina- 
tions be made within a few hours at the most. If the esti- 
mation of the dye must be delayed for some hours or days. 
the urine should be rendered distinctly acid, as the phtha- 
lein remains unchanged in acid solution. Just before mak- 
ing the colorimetric determination an excess of alkali is 
then added to elicit the full strength of the color. 

When urine is highly pigmented, error in the colorimet- 
ric readings may be lessened by making up a standard solu- 
tion containing urine. The error from this source is. how- 
ever, so small as to be negligible in most instances. 



CHAPTER II 

THE GASTRIC JUICE 

The gastric juice is obtained for analysis with the stom- 
ach tube, following the administration of a test breakfast 
or meal. The test breakfasts or meals are employed for 
the sake of simplicity and to obtain comparable conditions. 
It is because of the many unknown factors involved, such 
as the quality of food, the length of time it has remained 
in the stomach, the condition of the stomach before the 
food was taken, etc., that little dependence can be placed 
on the results of analysis of vomitus. 

Test Breakfasts. 

(1) Ewald's breakfast consists of 40 gm. of bread and 
400 c. c. of water or weak tea without sugar or cream. 

(2) Dock's breakfast is the same as the Ewald break- 
fast, except for the substitution of one shredded wheat bis- 
cuit for the bread. 

(3) Boas' breakfast is prepared by boiling one table- 
spoonful of oatmeal in 800 c. c. of water till the volume 
equals about 400 c. c. 

In this country Dock's breakfast is rapidly coming into 
use. This and the Boas breakfast possess a certain advan- 
tage over the Ewald breakfast, in that no lactic acid is con- 
tained in the food, a possible source of error when bread 
is used. Any of the breakfasts is allowed to remain in the 
stomach one hour, as a rule, at the end of which the stom- 
ach tube is introduced and the gastric contents evacuated. 
10 125 



126 TEST BREAKFASTS 

"With normal gastric motility the stomach yields 20 to 50 
c.c. one hour after a test breakfast (Boas). To eliminate 
the possibility of error through giving the breakfast to a 
patient whose stomach contains part of the previous meal, 
lavage may precede the breakfast, being performed prefer- 
ably an hour or so before giving the breakfast. In using 
the test breakfasts misinterpretation may follow if con- 
clusions are drawn from the results of a single meal. 

(4) The Fischer meal consists of an Ewald or Dock 
breakfast with three-quarters of a pound of finely chopped, 
lean beef, broiled and slightly seasoned. This meal, like 
the Eiegel dinner, excites the secretion of hydrochloric acid 
better than the breakfasts. It is usually allowed to remain 
in the stomach three hours. 

(5) The Riegel dinner is more appetizing than any of 
the preceding meals. It is composed of: 

One plate of meat broth. 

Beefsteak weighing 150 to 200 gm. (5 to 7 oz.). 

Mashed potatoes, 50 gm. (iy 2 oz.). 

One roll. 

Eiegel says: 1 "As a rule, I empty the stomach four 
hours after the meal, provided that other indications are 
not present that determine me to select some other time. 
If the stomach is found empty after four hours, I know 
that the motor power of the organ is good ; no conclusions, 
however, can be drawn in regard to its peptic powers. If 
the stomach is found empty after four hours, its contents 
should be withdrawn earlier the next day ; if, on the other 
hand, a large quantity of coarse and only half-digested 
morsels of food are found after four hours, the examina- 
tion on the next day should be made later. A single, exam- 

1 Eiegel, F. "Diseases of the Stomach" (edited by C. G-. Stockton). 
"Nothnagel's Practice."' Philadelphia and London, 1905, pp. 79 et seq. 



THE GASTRIC JUICE 127 

ination is never permissible." The dinner is usually given 
at the time of the midday meal. 

Other test meals have been proposed but are not very 
generally employed for purposes of gastric analysis. 1 



EXAMINATION OF THE FASTING STOMACH 

As the examination of the fasting stomach should pre- 
cede test meals, the results obtained may be considered be- 
fore passing to the examination of the gastric contents. 

The normal stomach empties itself in about seven hours. 
Passage of the stomach tube before breakfast should, there- 
fore, lead to the recovery of little fluid or none at all. Nor- 
mally, the amount rarely exceeds 50 c. c. (Emerson). When 
100 c. c. or more are obtained, there exists either a gastro- 
succorrhea (continuous secretion) or retention of the gas- 
tric contents (Boas). Normally or with hypersecretion, 
swallowed saliva or sputum may be seen in the fluid. With 
retention, food eaten the previous evening or several days 
before may be recognized; this should always be looked 
for, as it furnishes conclusive evidence of stagnation. The 
ease with which the food may be recognized will depend 
upon two factors: (1) the chemical composition of the gas- 
tric secretion, and (2) the nature of the food. With good 
acidity proteins may be well digested, whereas with a defi- 
ciency of acid they are little altered. Parts of food which 
resist the action of the gastric juice, such as the seeds of 
small fruit or berries, are easily detected. In fact, when 
defective motor power is suspected, it is a useful procedure 
to give raspberry jam or some similar preparation in the 
evening, and look for the seeds in the gastric contents or 

1 Riegel, F. "Diseases of the Stomach" (edited by C. G. Stockton). 
' ' Nothnagel 's Practice." Philadelphia and London, 1905, p. 79 et seq. 



'128 EXAMINATION OF GASTRIC CONTENTS 

lavage the following morning. At times excessive quan- 
tities of fluid are found in the fasting stomach. The nor- 
mal organ has a capacity of about 1,600 c. c. (Ewald) ; a 
stomach which can retain more than this quantity is di- 
lated. 

In addition to the points just enumerated, the fluid ob- 
tained from the fasting stomach should be subjected to 
the examination to be described for the gastric contents. 

MACROSCOPIC EXAMINATION OF THE GASTRIC 
CONTENTS 

Quantity.— In the examination of the gastric contents 
obtained one hour after a test breakfast, the quantity of 
fluid recovered is measured. Boas finds that the amount 
usually lies between 20 and 50 c. c. with normal gastric 
motility. Higher amounts, however, are certainly obtained 
in health at times ; 80 c. c. is not unusual. When 150 to 200 
c. c. are found in the stomach, hypomotility is quite defi- 
nitely indicated. A stomach which is repeatedly found 
empty one hour after a test breakfast has hypermotility, 
and it is then necessary to remove the contents after three- 
quarters or one-half hour. 

Odor.— The normal gastric contents are practically 
odorless. In disease the odor may be sour or rancid (ace- 
tic acid, butyric acid, etc.), putrid, fecal, etc. The odor of 
drugs should also be looked for. 

Mucus.— The presence of an excess of mucus is most 
easily detected by pouring the gastric juice from one re- 
ceptacle to another. If the amount be abnormal, the con- 
dition is at once recognized. Mucus from the respiratory 
passages floats because of the bubbles contained in it. 
From the pharynx and esophagus there may be a consid- 



THE GASTRIC JUICE 129 

erable quantity of mucus secreted during the passage of 
the stomach tube. It runs along the side of the tube, and 
is not aspirated- through the tube, as in the case of true 
gastric mucus or swallowed sputum. 

Color.— Normally the gastric secretion is practically 
colorless. The regurgitation of bile from the duodenum 
may impart a deep yellow or green color, the intensity 
depending on the relative proportion of bile. Blood, when 
fresh, is characteristic in appearance; if it has remained 
in the stomach long enough to undergo change, the bright 
red color is lost, and is replaced by a dark brown, pro- 
ducing in many instances the so-called "coffee-ground" ap- 
pearance. The color of the gastric juice may also be al- 
tered by food or drugs. 

Food.— The state of digestion of the food is of great im- 
portance. After the usual test breakfasts, carbohydrate 
forms the bulk of the food ingested. The alterations found 
are due chiefly to ptyalin of the saliva. With hyperacid- 
ity this enzyme is quickly destroyed, with a consequent in- 
hibition or arrest of amylolysis. After a mixed meal, such 
as the Riegel dinner, more information may be gained by 
inspection of the gastric contents. The appearances are 
well described by Eiegel. 1 "In some cases a very fine, uni- 
form, mushy liquid mass is seen that contains no coarse 
elements at all ; in others, again, a mass of food containing 
many coarse pieces of meat that look as if they had just 
been swallowed; in addition, there is frequently an abun- 
dant admixture of mucus. In some cases there is so much 
mucus that the food looks like a tough mass and passes 
through the sound with difficulty, and is very difficult to 
filter. In other cases there is a large quantity of fluid con- 
tents that forms three layers when kept in a glass vessel; 

1 Riegel, F. Loc. cit., p. 86. 



130 EXAMINATION OF GASTRIC CONTENTS 

at the bottom is seen a layer consisting of fine remnants of 
amylaceous material; above this a large layer of cloudy 
fluid, and on the top a foamy layer. If the latter is pres- 
ent it may be considered evidence of gaseous fermenta- 
tion. This consistency of the stomach contents is found 
chiefly in cases in which there is stagnation or in which 
there is motor insufficiency . . . usually in' cases in 
which there is an abundant quantity of free hydrochloric 
acid. ... If the food remnants obtained from the 
stomach in different diseases are compared, the great sig- 
nificance of macroscopic inspection will be understood. In 
many instances this method alone will give us diagnostic 
points which we would otherwise obtain only by compli- 
cated chemical examinations. There are cases, for in- 
stance, in which the stomach contents do not give any of 
the reactions for free hydrochloric acid. This shows that 
there is a deficit in the stomach. Sometimes, however, 
when free hydrochloric acid is absent, we find only a rela- 
tively small amount of finely distributed food residue ; at 
other times we may see larger quantities of coarse food 
particles. If we limit ourselves to examining the filtrate 
in both these cases for free hydrochloric acid, we will prob- 
ably consider that the two are alike, and, as a matter of 
fact, they are alike in regard to their free hydrochloric 
acid, for in neither do we see a formation of free hydro- 
chloric acid. If, however, we consider the quantity and 
the appearance of the stomach contents in both, we shall 
see that in the first case the peptic power is better than in 
the second. The first case is functionally nearly normal, 
for all the albumin has been digested; at the same time 
there was no residue of free hydrochloric acid. In the sec- 
ond case it is different; here the production of acid was 
subnormal, as shown by the disturbed digestion of meat. 



THE GASTRIC JUICE 131 

If this case is more carefully examined, it will be found 
that the deficit of hydrochloric acid is large, whereas in the 
first case it is small. In this way macroscopic examination 
frequently gives us a clear picture of disturbances of func- 
tion. . . . " 

The careful macroscopic analysis of the gastric con- 
tents, it is evident, is of the greatest value. 



CHEMICAL EXAMINATION OF THE GASTRIC CONTENTS 

Reaction.— The reaction of the gastric contents is tested 
with litmus paper. It is usually acid. An alkaline or neu- 
tral fluid may be obtained. 

Hydkochlokic Acid 

Hydrochloric acid is the most important chemical con- 
stituent of the gastric juice from the clinical standpoint. 
Normally it is present in excess, i. e., a test for free hydro- 
chloric acid is always obtained. 

Qualitative Tests for Free Hydrochloric Acid 

(1) Von den Velden's Methyl Violet Test.— Add a few 
drops of a saturated aqueous solution of methyl violet to 
a test tube nearly filled with water. The dilute solution of 
the dye should be transparent and violet or purple in color. 
It is divided equally in two test tubes. To the one add an 
equal quantity or less of gastric juice, to the other an equal 
volume of water. Free hydrochloric acid is indicated by a 
change in color from violet to blue, the portion to which 
water alone is added serving as a control. The test is said 
to indicate 0.025 per cent, of free hydrochloric acid. Ac- 



132 EXAMINATION OF GASTEIC CONTENTS 

cording to Biegel, the test is especially valuable, since, 
when it is positive, it means that there is sufficient free acid 
for protein digestion. 

A second method of performing the test, which is use- 
ful when the amount of gastric juice at one's disposal is 
small, consists in spreading a thin layer of the dilute methyl 
violet solution in a porcelain plate, and then placing a drop 
of gastric juice in contact with it. Where the two fluids 
run together, the violet color is changed to blue in the pres- 
ence of free acid. 

Lactic acid does not interfere with the methyl violet re- 
action, since it is given only by 0.4 per cent, or stronger 
solutions, which never occur in the stomach. 

(2) Giinzberg's Test.— This is the standard test for free 
hydrochloric acid. It is positive only in the presence of a 
free mineral acid. 

Eeagent : 

Phloroglucin 2.0 gm. 

Vanillin 1.0 gm. 

Alcohol, absolute 30.0 c. c. 

Dissolve and keep in a brown bottle, tightly 
stoppered. As the reagent does not keep well, 
it is advisable to make small quantities, so 
that it may be renewed every few months. It 
is well to test the reagent from time to time 
with dilute hydrochloric acid to prove its re- 
liability. 

A few drops of the reagent are evaporated to dryness 
in a porcelain dish by warming gently over a Bunsen bur- 
ner. A drop of gastric contents is brought in contact with 



THE GASTRIC JUICE 133 

the yellowish-brown stain left by the reagent, and is evap- 
orated. If free hydrochloric acid is present, an intense red 
color develops, where the reagent and gastric juice have 
mixed. Instead of evaporating the reagent and gastric 
juice separately, equal quantities of the two may be mixed 
(one or two drops of each) and evaporated, when the color 
change appears. 

The evaporation must be performed with great care. It 
is easy to burn the reagent by overheating; the test then 
fails, even though there be an abundance of free acid pres- 
ent. The degree of heat may be tested by touching the 
bottom of the porcelain dish with the finger. The dish is 
held in the flame a second, removed, tested ; this procedure, 
repeated at intervals, accomplishes the desired result with 
a little practice. Blowing on the specimen when it is re- 
moved from the flame hastens the evaporation, and at the 
same time lowers the temperature. A safer method of 
evaporation is the use of a water bath. 

The test is sensitive to free hydrochloric acid in 0.01 
per cent, solution. It is specific in the sense that a positive 
reaction is only obtained with free mineral acid; organic 
acids do not give the test. 

(3) Tropeolin Test.— A saturated alcoholic solution of 
tropeolin 00 is prepared. Three to four drops of this re- 
agent and a like quantity of the gastric juice are spread 
over the surface of a porcelain dish, and carefully evapo- 
rated to dryness. In the presence of free .acid the color be- 
comes violet or blue. The test is less sensitive than either 
of the preceding tests. It is positive with free hydrochloric 
acid in a dilution of 0.03 per cent. Lactic acid solutions 
of 0.24 per cent, or stronger give the reaction (Ewald) ; in 
the stomach it is doubtful whether lactic acid ever occurs 
in sufficient concentration to give the test. 



134 EXAMINATION OF GASTRIC CONTENTS 

In place of the concentrated alcoholic solution of trope- 
olin 00, Eiegel recommends a saturated aqueous solution. 

(4) Congo-paper Test.— Filter paper is saturated with 
a concentrated aqueous solution of Congo-red, and allowed 
to dry. It is then cut into narrow strips. A piece of the 
paper is moistened with the stomach contents. Free hy- 
drochloric acid turns the paper deep blue; lactic acid pro- 
duces a much less intense blue. The test is fairly delicate, 
but with very dilute solutions of hydrochloric acid the color 
change is very slight and rather difficult to interpret. Lac- 
tic acid is never found in sufficient concentration to lead to 
difficulty, according to Eiegel. 

(5) Topfer's Test.— One drop of 0.5 per cent, alcoholic 
solution of dimethylamidoazobenzol is added to a few c. c. 
of gastric juice. Free hydrochloric acid produces a bright 
red color. Organic acids also cause a color reaction, but 
the color is less brilliant — more of a brick red. The reac- 
tion is, therefore, not specific, and is the least reliable of 
the tests. 

Of the tests for free hydrochloric acid, the Giinzberg 
test is the most delicate and at the same time the most re- 
liable. It is a good routine test, and in any case should be 
employed wherever doubt exists. 

In certain instances where it is desirable to have infor- 
mation regarding the acid secretion of the stomach, contra- 
indications to the passage of the stomach tube exist. In 
such case Sahli's desmoid test may be used. 

(6) Sahli's Desmoid Test. 1 — This is a test for free hy- 
drochloric acid. It is based on the fact that raw catgut is 
soluble in hydrochloric acid-pepsin, insoluble in pancreatic 
and intestinal juices. 

1 Boggs, T. E. "Sahli's desmoid reaction in gastric diagnosis." Bull. 
Johns Hopkins Hosp., 1906, XVII, 313. 



THE GASTRIC JUICE 135 

Pills of the following formula are prepared: 

Methylene blue 0.05 gm. 

Iodoform 0.1 gm. 

Ext. glycyrrhiz q. s. 

The pills should not exceed 3 or 4 mm. in diameter. 
The iodoform may be omitted. The pill is placed in the 
center of a square of thin rubber dam, such as dentists 
use. The rubber is stretched and twisted about the pill. 
The twisted neck is then tied with three turns of raw No. 
00 catgut, previously soaked in cold water till soft. Now 
trim the rubber so that a free edge of about 3 mm. width 
remains beyond the ligature. The cut edges of the rubber 
must not cohere, inclosing air, for the pill must sink in 
water, and it must be watertight. 

A pill prepared as described is given to the patient with 
his midday meal, and the urine, collected 5, 7, 18, and 20 
hours afterward, is examined for the presence of methylene 
blue, iodin, or both. In the absence of the greenish color 
of methylene blue, the urine should be boiled with one-fifth 
volume of glacial acetic acid. If the chromogen of methy- 
lene blue exists in the urine, the color will then appear. 
Iodin may be looked for with Obermayer's test for indican 
(p. 27). If methylene blue appears in the urine within 
twenty hours after the administration of the pill, the test 
is considered positive. 

A positive test shows that there is sufficient free hydro- 
chloric acid secreted in the stomach to permit of digestion 
of the raw catgut and liberate the pill from its rubber 
capsule. If the gastric juice fails to digest the catgut, the 
pill passes into the intestines and is evacuated. The test 
is, therefore, one for free hydrochloric acid. As it is given 
with a regular meal, it encounters the optimal conditions 



136 EXAMINATION OF GASTRIC CONTENTS 

for acid secretion. The test is thus a useful adjuvant to 
the usual gastric analyses in certain cases of anacidity. 

Organic Acids.— When free hydrochloric acid is mark- 
edly diminished or entirely lacking, tests for organic acids 
should be made. With normal hydrochloric acid values, 
lactic acid fermentation does not occur. The tests for or- 
ganic acids are described on pages 141-142. 

Quantitative Determination of Gastric Acidity 

In the quantitative analysis of the gastric juice the 
amount of free hydrochloric acid and of total acidity and 
the extent of the hydrochloric acid deficit are of importance 
clinically. Very little of diagnostic value has resulted from 
estimation of the loosely combined hydrochloric acid, i. e., 
hydrochloric acid in protein combination. 

Topfer's Method for Free Hydrochloric Acid.— This is 
the method generally employed, since it is quickly carried 
out and is sufficiently accurate for clinical purposes. 

A drop of 0.5 per cent, alcoholic solution of dimethyl- 
amidoazobenzol is added to 10 c. c. 1 of filtered gastric con- 
tents, placed in a porcelain dish or in a beaker resting on 
a sheet of white paper for a background. The gastric juice 
should be measured accurately with a pipette. In the pres- 
ence of free hydrochloric acid, the addition of the drop of 
indicator produces a brilliant red color in the liquid. From 
a burette graduated in tenths of a cubic centimeter, tenth 
normal sodium hydrate is run into the mixture, a few drops 
at a time, with constant stirring, till the red color entirely 
disappears. This is the end reaction. The quantity of 
tenth normal hydrate required to neutralize the acid in 
10 c. c. of the gastric contents is then read from the burette. 

1 If the quantity of gastric contents obtained is small the titration is made 
with 5 c. c, with a corresponding correction in the final calculation. 



THE GASTRIC JUICE 137 

The result is usually expressed as "acidity per cent./ 7 
i. e., the number of cubic centimeters of tenth normal alkali 
which would be required to neutralize the free acid in 100 
c. c.of gastric contents. Since 10 c. c. were taken, the quan- 
tity of alkali used, multiplied by 10, gives the desired re- 
sult. Normally free hydrochloric acid varies between 20 
and 40. The amount of hydrochloric acid may be calcu- 
lated. One c. c. of tenth normal alkali is equivalent to 
0.00365 gm. HC1. 

If the amount of gastric juice is small, the same sample 
may be employed for the determination of total acidity. 
A drop of phenolphthalein is added and the titration con- 
tinued. The alkali used in neutralizing the free hydrochlo- 
ric acid must, of course, be included in the total acidity. 

Dimethylamidoazobenzol is not the ideal indicator, since 
it reacts with organic acids and acid salts as well as with 
mineral acids. The results obtained with it are, therefore, 
too high; they do not represent absolute values. Never- 
theless, the method fulfills all clinical needs, since the error 
introduced is relatively so small that it does not vitiate 
the results for diagnostic purposes. 

Other Indicators.— In place of dimethylamidoazobenzol 
Giinzberg's reagent and Congo-red are frequently employed 
as indicators in the titration of free hydrochloric acid. 

Giinzberg's reagent may be used in several ways. As 
the titration progresses, a small drop of the gastric juice 
is removed with the stirring rod from time to time, and 
placed on the evaporated Giinzberg's reagent. The drop is 
evaporated, and the red color appears at the margin as 
long as free acid exists. A second procedure consists in 
the addition of 25 to 30 drops of Giinzberg's reagent to the 
gastric juice, and then at intervals the removal of a minute 
drop, which is evaporated in the usual manner. The glass 



138 EXAMINATION OF GASTRIC CONTENTS 

stirring rod itself may be gently warmed till the fluid cling- 
ing to it is evaporated; it is then examined for the red 
color. The disadvantage in these procedures is that a 
small quantity of the gastric contents is lost with each test 
for free acid, so that the result is slightly low. Compara- 
tive titrations with Giinz berg's reagent and dimethylamido- 
azobenzol will show less free acid, as a rule, when Gunz- 
berg's reagent is used; occasionally the values are alike. 

Congo-red paper may also serve as the indicator in the 
titration of free hydrochloric acid. It is very convenient 
for night work. The tenth normal alkali is added to the 
gastric juice until a small drop placed on Congo-red paper 
no longer produces a blue color. As a control the paper 
should be moistened with distilled water, for the red color 
becomes somewhat darker when moistened. The results 
are usually intermediate between those obtained with Giinz- 
berg's reagent and those with dimethylamidoazobenzol. 

Titration of Total Acidity. — The total acidity comprises 
free hydrochloric acid, loosely combined hydrochloric acid 
(i. e., in combination with protein), acid salts, and organic 
acids, such as lactic, butyric, and aminoacids, when pres- 
ent. Its quantity is determined by titration with tenth 
normal alkali, using phenolphthalein as the indicator. 

With a pipette measure 10 c. c. (or 5 c. c.) of filtered 
gastric contents into a porcelain dish or Erlenmeyer flask 
placed on a sheet of white paper, and add one or two drops 
of 0.5 per cent, alcoholic solution of phenolphthalein as in- 
dicator. In an acid medium it is colorless, but it becomes 
pink as soon as all the acid is neutralized, leaving a slight 
excess of alkali. Tenth normal sodium hydrate is added 
from a burette under constant stirring, until the whole mix- 
ture takes on a faint pink color, which persists. The num- 
ber of c. c. of alkali used, multiplied by 10 (or by 20 in 






THE GASTRIC JUICE 139 

case 5 c. c. of gastric contents were taken), gives the total 
acidity per cent. Normally this varies between 40 and 
60 or 70. 

The results obtained are again only approximately cor- 
rect, being too high as a rule. For diagnostic purposes the 
method is practicable. 

The Hydrochloric Acid Deficit 

A deficit in hydrochloric acid occurs whenever the gas- 
tric mucosa secretes so small a quantity of hydrochloric 
acid that there is not merely an absence of free acid, but 
an excess of bodies capable of binding or uniting with it. 
Such bodies are chiefly proteins and their end-products, 
peptids, and the aminoacids. If peptic digestion of the 
proteins alone occurs, the aminoacids are not concerned 
in the production of a deficit in hydrochloric acid, since 
pepsin is unable to carry the hydrolysis of the protein 
molecule to the aminoacid stage. But, when trypsin is re- 
gurgitated into the stomach, or when the proteolytic en- 
zyme of a malignant neoplasm is secreted into the stomach, 
aminoacids may be abundant in the stomach contents ; they 
may also be the result of bacterial decomposition, though 
probably not frequently. The presence of aminoacids is of 
significance in two directions in the quantitative analysis 
of the gastric contents, as Fischer 1 has pointed out. Pep- 
sin converts the proteins into peptids, which react alka- 
line toward litmus ; when united with hydrochloric acid the 
reaction is reversed. The hydrolysis of the peptids into 
their constituent aminoacids alters the conditions. The 
latter can bind hydrochloric acid and at the same time 
carboxyl groups are liberated. The result is that the total 

1 Fischer, H. ' ' Zur Kenntnis des carcinomatoseu Mageninhaltes. ' ' 
Deutsch. Archiv f. Min. Med., 1908, XCIII, 98. 




140 EXAMINATION OF GASTRIC CONTENTS 

acidity is increased, while the free hydrochloric diminishes. 
With an excess of aminoacids it is then necessary to add 
more or less hydrochloric acid before a reaction for free 
acid is obtained. Factors which play a less important role 
in the production of an acid deficit are alkalies introduced 
with the food or secreted, possibly, in disease. 

It is unnecessary to remark that only those specimens 
of gastric juice which fail to react to Gunzberg's reagent 
for free hydrochloric acid are suitable for the determina- 
tion of a deficit in acid. 

The method of determining the deficit in free hydro- 
chloric acid is as follows : From a burette add tenth nor- 
mal hydrochloric acid to 5 or 10 c. c. of the gastric contents 
with constant stirring, until a test for free hydrochloric 
acid is obtained. For this purpose the Giinzberg test is 
to be preferred. Dimethylamidoazobenzol is not well 
adapted to the titration, since organic acids which are of- 
ten present react with it; Congo-red paper gives more sat- 
isfactory results than dimethylamidoazobenzol. The extent 
of the deficit may be expressed as "deficit per cent.", — 
the usual way; the number of cubic centimeters of tenth 
normal hydrochloric acid which would be required for 100 
c. c. of gastric contents is calculated. Or the deficit may 
be expressed in terms of hydrochloric acid, calculated for 
100 c. c. of stomach contents. 

Oeganic Acids 
Lactic Acid 

Of the organic acids which may be present in the stom- 
ach contents in disease, lactic acid is the most important 
and is the only one tested for in the usual routine exam- 



THE GASTRIC JUICE 141 

ination. It is odorless. Lactic acid is the result of fer- 
mentation of the gastric contents. The fermentation oc- 
curs only in the absence or very marked decrease of free 
hydrochloric acid. When many Oppler-Boas bacilli are 
present in the gastric contents, lactic acid is usually found, 
though the converse is not true. Lactic acid almost al- 
ways means stasis of the gastric contents ; it is not found 
in anacidity, where the motor power of the stomach is nor- 
mal. Quantitative estimation of lactic acid has not been 
found of value in diagnosis. 

Qualitative Tests for Lactic Acid. — (1) Ueffelmann's 
Test. — To 15 or 20 c. c. of 1 per cent, aqueous carbolic acid 
in a test tube, 10 per cent, ferric chlorid solution is added 
till an amethyst color is produced; usually 1 to 2 drops 
suffice. If necessary, the solution is diluted till it is trans- 
parent, and is then divided equally between three tubes. 
To the first a few drops of the filtered gastric contents are 
added, to the second a like quantity of distilled water to 
serve as a control, and to the third the same amount of 
dilute lactic acid solution for comparison with tube one. 
A yellowish-green (canary yellow) color denotes lactic acid 
or its salts. A similar color reaction may also be given by 
oxalic, citric, and tartaric acids, by alcohol and dextrose, 
but these substances can usually be excluded after an 
Ewald or Dock breakfast. 

To avoid error from disturbing bodies, it has been rec- 
ommended to extract the gastric contents with about ten 
volumes of ether, which is then evaporated; the residue is 
dissolved in water, to which the test is applied. 

(2) Stkauss' Test. — To avoid the sources of error in 
the preceding test, Strauss employs a specially devised 
separating funnel, which is used to extract the gastric con- 
tents. Above the glass stopcock there are two marks which 
li 



142 EXAMINATION OF GASTRIC CONTENTS 

correspond to 5 c. c. and 25 c. c. The gastric contents are 
added to the mark 5, and then ether is poured to the mark 
25. The two fluids are mixed thoroughly by shaking, and 
after they have separated the gastric contents are allowed 
to escape. Distilled water is then added till the ether again 
rises to the mark 25. After the addition of one drop of 10 
per cent, ferric chlorid solution, shake vigorously, and wait 
for the fluids to separate. In the presence of lactic acid a 
greenish-yellow color is imparted to the watery layer. The 
extraction with ether separates the lactic acid from the in- 
terfering bodies. If lactic acid is combined with protein, 
the test may be negative; but the lactic acid may be freed 
by the addition of dilute hydrochloric acid, until a test for 
the latter is given with Congo paper. The test now be- 
comes positive. 

(3) Kellixg's Test. — A small portion of the gastric 
contents is diluted with 10 to 20 volumes of distilled water. 
A second test tube is filled with the same quantity of water 
alone. To each tube add one drop of 10 per cent, ferric 
chlorid. Lactic acid causes a canary-yellow color. Dilu- 
tions of 1:10,000 to 1:15,000 may give a positive reaction. 
The second tube, containing water and ferric chlorid, 
serves as a control. As in Uffelmann's test, the color is 
often perceived most easily by looking down into the test 
tube, which is held on a white background. 

Butyric Acid 

Butyric acid fermentation may take place in the pres- 
ence of considerable quantities of free hydrochloric acid. 
The odor of butyric acid, resembling that of rancid butter, 
is characteristic. Boiling the gastric contents accentuates 
the odor ; if a piece of moistened blue litmus paper be held 



THE GASTRIC JUICE 143 

in the mouth of the test tube, the volatile acid reddens it 
as it escapes during the boiling. Butyric acid also has the 
peculiar property of separating as a drop of oil on the ad- 
dition of a small piece of calcium chlorid. 

Acetic acid, like butyric acid, may be recognized by its 
odor if present in sufficient concentration. Acetic acid, 
after careful neutralization with sodium hydrate, with the 
formation of sodium acetate, gives a blood-red color on the 
addition of a drop of ferric chlorid. 

Gastkic Fekments 

Normally pepsin and rennin, or their zymogens, are 
constituents of the gastric juice. Alterations in the en- 
zymes in disease are much less frequent and less striking 
than those occurring in the hydrochloric acid. Whenever 
the latter is present, it is practically always the case that 
pepsin is also found. With absence of free hydrochloric 
acid, tests for the enzymes should be made. Quantitative 
determination of pepsin has not proved to be sufficiently 
valuable to warrant its inclusion in the usual routine gas- 
tric examinations. 

Pepsin 

Qualitative Test for Pepsin.— Discs of coagulated egg 
albumin, ca. 1.5 mm. thick and 5 to 10 mm. in diameter, 
are cut with a cork-borer or goose-quill. They may be 
preserved in glycerin, but should be washed in water im- 
mediately before use to remove the excess of glycerin. A 
disc of the coagulated albumin is placed in a few c. c. of 
gastric contents, and, if necessary, dilute hydrochloric acid 
is added, till Congo paper gives a test for free acid. The 
material is then placed in an incubator at 37° C. (or in 



144 EXAMINATION OF GASTRIC CONTENTS 

the vest pocket). In one-half to one hour the albnmin 
should be digested. 

Fibrin, usually obtained from ox blood and preserved 
in glycerin, may be substituted for the coagulated egg al- 
bumin. 

Quantitative Methods.— Quantitative methods for pep- 
sin may occasionally be desirable. Several have been pro- 
posed within the last few years. The results given by each 
are relative, not absolute, values. 

Mette's Method as Modified by Xierexsteix axd 
Schiff. 1 — Capillary glass tubes, 1 to 2 mm. in diameter 
and 20 to 30 cm. in length, are filled with egg albumin by 
suction, the ends plugged with bread crumbs, and the tubes 
then placed in boiling water for five minutes. They are 
then sealed with paraffin or sealing wax. Bubbles appear 
in the albumin, but are no longer seen after three days, 
when the tubes are ready for use. If the albumin retracts 
from the wall of the tube, it should not be used for the test. 

Method. — One c. c. of filtered gastric contents is diluted 
with 15 c. c. of twentieth normal hydrochloric acid. AVith a 
file or glass scissors, cut off about 2 cm. of the capillary 
tube, and place two such pieces in the diluted gastric con- 
tents. The test tube is then corked and placed in an incu- 
bator at 37° C. for twenty-four hours. At the end of this 
time the tubes are removed and the amount of digestion 
of albumin in the four ends of the capillary tubes is meas- 
ured in tenths of a millimeter, a hand lens being useful for 
this purpose. An average of the four readings is taken. 
The square of this number represents the number of units 
of pepsin present in the diluted gastric contents. Multi- 

1 Farr, C. B., and Goodman, E. H. "The clinical value of the quantitative 
estimation of pepsin, with special reference to the Mette and ricin methods." 
Arch. Int. Med., 1908, I, 648. 



THE GASTRIC JUICE 145 

plying this by 16 gives the value for the undiluted speci- 
men. 

Since the albumin from different eggs may react differ- 
ently, and since the length of time the albumin is boiled 
affects its digestibility, the method can be relied upon only 
to show rather wide variations in pepsin. 

According to Cowie, 1 the tubes need not remain in the 
incubator twenty-four hours. He finds that the amount of 
digestion varies directly as the time. He derives the fol- 
lowing formula for calculating the digestion : 

If A=the amount of egg white digested, 

B— the time the tubes remain in the incubator, 
C=the required time for the end reaction, 
X=the peptic value of the fluid tested, or the esti- 
mated value in millimeters, 

then it will be found that X= AxC 

B 

Rennin 

Eennin, the enzyme responsible for the coagulation of 
milk, may exert its characteristic action in the absence of 
hydrochloric acid. Rennin zymogen, inactive in itself, is 
converted into rennin by acid. The zymogen is much more 
resistant to alkalies than rennin. 

Qualitative Test for Rennin.— Three to 5 drops of fil- 
tered gastric contents are added to 5 to 10 c. c. of raw, 
amphoteric, or neutral milk. After mixing thoroughly, the 
fluid is placed in the incubator for 15 to 20 minutes. The 
presence of rennin is shown by the curdling of the milk, 
provided its reaction remains neutral or amphoteric. If 

1 Cowie, D. M. "A rapid procedure for the estimation of the peptic value 
of stomach fluid by means of the Mette method. ' ' The Phys. fy Surg., Detroit 
and Ann Arbor, 1904, XXIV, 118. 



146 EXAMINATION OF GASTRIC CONTEXTS 

the reaction has become acid, it is probable that fermenta- 
tion or "souring" of the milk is the cause of the curdling. 
Eiegel recommends that equal parts of milk and gastric 
contents be taken. The latter is first neutralized with tenth 
normal alkali. Curdling should occur within 15 to 30 min- 
utes. Acid reaction of the milk after incubation invalidates 
the test, as in the preceding instance. 

Rennin Zymogen 

Eiegel gives the following method for detecting rennin 
zymogen : Ten c. c. of the gastric contents are rendered 
alkaline with tenth normal sodium hydrate to inactivate 
rennin. Then add 10 c. c. of fresh, neutral, or amphoteric 
milk and 3 to 5 c. c. of 1 to 2 per cent, calcium chlorid solu- 
tion, and place the mixture in the incubator at body tem- 
perature. If the zymogen is present, casein is precipitated 
within a few minutes. 



Pathological Enzyme in the Gastric Contents 

It has been demonstrated that malignant neoplasms 
contain a proteolytic enzyme which, unlike pepsin, is ca- 
pable of splitting proteins into their constituent arnino- 
acids. This fact has recently been utilized by Xeubauer 
and Fischer 1 in devising a test for cancer of the stomach. 
The test has been used with varying success by a number 
of observers. The test is certainly positive in many cases 
of anacidity unassociated with malignant disease of the 
stomach. The explanation of the occurrence of the positive 

1 Xeubauer. O.. and Fischer. H. ' ' TTeber das Vorkommen eines peptid- 
spalrenden Femientes im eareinomatosen Mageninhalt und seine diagnostisehe 
Bedeutung." DeutscJi. Archiv f. Win. Med.. 1909. XC'VII. 499. 



THE GASTRIC JUICE 147 

test in these cases was not clear, until Warfield 1 demon- 
strated the presence of a peptid-splitting enzyme in nor- 
mal saliva. If the saliva is acid or comes in contact with 
an acid fluid (0.05 per cent, plus), the salivary peptid-split- 
ting enzyme is destroyed. Warfield 's observations on the 
saliva have been confirmed by Koelker, 2 and would seem to 
invalidate the test of Neubauer and Fischer. As the pres- 
ence of a peptid-splitting enzyme in carcinomata and 
sarcomata is well established, it seems probable that not 
all the positive results are attributable to swallowed 
saliva. The test, therefore, must be interpreted Avith ex- 
treme caution in its present form. That there are still pos- 
sibilities of altering the technique, so that more reliable 
results may be obtained with it, must be admitted, and 
largely for this reason it is included as given by Neubauer 
and Fischer. 

Method. — An Ewald or Dock breakfast is removed after 
it has remained in the stomach one-half to three-quarters 
of an hour. The filtered juice is then tested for the pres- 
ence of bile (and consequently, in all probability, for pan- 
creatic juice) with dilute tincture of iodin by layering it 
above the fluid (a green line at the juncture of the two 
fluids denotes bile pigment), and for blood by means of 
the guiac test. It is obligatory to exclude both pancreatic 
juice and blood, since each contains an enzyme capable of 
hydrolizing polypeptids to aminoacids. If pancreatic juice 
and blood are lacking, the test for the cancerous peptid- 
splitting ferment is proceeded with. The gastric juice is 
tested for preformed tryptophan, as described below, and, 
if none is found, one adds about 10 c. c. of gastric contents 

1 Warfield, L. M. "A peptid-splitting ferment in the saliva." Bull. Johns 
Hopkins Hosp., 1911, XXII, 150. 

2 Koelker, A. H. "Ueber ein Dipeptid- und Tripeptid-spaltendes Enzym 
des Speichels. ; ' Zeitschr. /. physiol. Chem., 1911, LXXVI, 27. 



145 EXAMINATION OF GASTRIC CONTENTS 

to 1 : giycyltryptophan; 1 the mixture is then covered 

with tolnol to prevent bacterial growth, and is placed in 
the thermostat at o7 C. for twenty-four hours. A rtion 
of the fluid is then removed with a pipette, and placed in 
a clean test tube. It is tested for tryptophan, the presence 
of which indicates the existence of a peptid-splitting fer- 
ment, in the following manner: Two to 3 e. : ~ v.: 1 : . 
acidified with a few drops of 3 per cent, acetic acid. Bro- 
min vapor is allow | -ettle in the test tube till a slight 
brownish tint is visible in the upper part of the tube; the 
vapor may be introduced better by means of a 10-c. c. pi- 
re armed with a rubber bulb. The contents of the tube 
are shaken. If a rose color develops free tryptophan is 
present, and the test ie sitive. If the rose color fails to 
appear bromiu is added as before. Unless the color ap- 
pears the procedure > ited until the fluid in the test 
tube shows a light yellow color, which indie:— an excess 
of broniin. An early excess of brornin is to be avoided. 
since the color u then sc evanescent that it may 
entirely es : | b notice. Instead of bromiu vapor, one-tenth 
rated aqueous solution of calcium chlorate may be 
The color reaction is the same, and an excess of the 
reagent is again to be avoided, as the color may be over- 
looked. 

Mucus 

Mucus is always present in the gastric juice, though in 
very small amount under normal conditions. When pres- 

1 Giycyltryptophan is a dipeprid, which has been plaeed upon the market 
in small bottles under the name ' ' Fermentdiagnosrikum. ' ' Eaeh bottle con- 
tains enough giycyltryptophan, with toluol added, for one test. It may also be 
had from the makers. KaHe & Co., Biebrieh am Rhedn. Germany, in bulk, and 
this is to be preferred because of the great saving in eost. In using the small 
bottles gastric contents are added to the line marked on the bottle, which is 
then stoppered and plaeed in the incubator. 



THE GASTKIC -JUICE 149 

ent in excess, the characteristic ropy or stringy quality of 
the fluid is seen on pouring it from one vessel to another. 
Microscopically, small snail-like masses of mucus may be 
seen. Mucus usually contains epithelial or pus cells, if 
the latter are present in the stomach contents ; with normal 
acidity digestion may leave only the nuclei. Mucus from 
the bronchi, pharynx, or esophagus is often observed in 
the gastric contents. Bronchial mucus usually contains 
bubbles, which cause it to float. Microscopic examination 
reveals alveolar cells, often laden with coal dust, and an 
abundance of pus cells frequently; an absence of food 
particles is often noted. The mucus from esophagus and 
pharynx, which is generally formed in abundance during 
the passage of the tube, runs out along the side of the tube. 
It should not be allowed to mix with the material obtained 
from the stomach. 

Anacidity often causes an apparent increase in mucus, 
even though there is no overproduction, since that which 
is formed is not normally digested, and, furthermore, it 
swells to an unusual degree. 

MICROSCOPIC EXAMINATION OF THE GASTRIC 
CONTENTS 

Normally one finds, after a test breakfast, only isolated 
bacteria, a few desquamated epithelial cells, many starch 
granules, a few fat droplets, possibly a few yeast cells (not 
budding), an occasional leukocyte at times, and small par- 
ticles of mucus. The fresh specimen should be employed 
for examination, which is made with the dry objectives. 

Starch granules are conspicuous. When well preserved, 
laminations are indicated by the concentric lines. If diges- 
tion has not proceeded too far, the starch granules are 



150 MICROSCOPIC EXAMINATION 

stained deep blue on the addition of a drop of Lugol's 
iodin solution. 

Fat droplets are recognized by their appearance and 
staining reactions (see p. 85). 

Blood corpuscles, when present in the gastric juice, are 
often too greatly damaged to be recognized microscopically. 
TTith a recent hemorrhage they may, however, present a 
characteristic appearance, particularly if enough blood has 
been shed to completely bind the free acid. Chemically, 
blood is detected more frequently. The guiac or benzidin 
tests are usually employed as preliminary tests. (For the 
technique of the chemical tests see the sections on the urine 
and feces.) 

Pus cells, when well preserved, differ in no way from 
those observed elsewhere. Generally the protoplasm of 
the cells has been digested, leaving only the naked poly- 
morphous nuclei. 

Eosinophilic leukocytes are a rare finding in the gastric 
contents. 1 

Microorganisms may exist in the stomach in large num- 
bers in disease. Normally their growth is prevented by the 
free hydrochloric acid and the rapid emptying of the organ. 

Yeasts are introduced into the stomach in small num- 
ber with the food, but they exhibit no sign of germination. 
In disease, on the other hand, often in the presence of con- 
siderable free hydrochloric acid, an active growth of yeasts 
may be found. Large colonies may be observed. The cells 
are oval bodies, smaller than a red corpuscle, which have 
a greenish, glistening appearance when seen with strong 
illumination. Characteristic budding forms — three or four 
cells linked together, with a progressive diminution in size 

1 Moaeanin. S. "Ueber das Vorkommen von eosinophilen Zellen im Ma- 
gensafte bei Achylia gastrica. " Wiener lclin. Wclinsclir., 1911, XXIV, 1335. 



THE GASTRIC JUICE 151 

— are common. They may be distinguished with the low 
power, and are easily recognized with the high power, dry 
objectives. 

Sarcince are found in the form of bales or packages, or 
as irregular masses of cells. Like yeasts, they are abun- 
dant in the stomach only in disease. • Two sizes, large and 
small, are met with, and the significance of each is the 
same. They are slightly brownish, and can often be found 
most easily with the low power. They are usually associ- 
ated with stasis of benign origin. 

Oppler-Boas bacilli are capable of producing lactic acid 
fermentation, and thus it happens that their growth in the 
stomach practically always results in the simultaneous 
presence of lactic acid in the stomach contents. The bacilli 
are characterized by their great size and lack of motility.^ 
They are long and have a tendency to grow in chains, which 
may at times extend across the field of the microscope. To 
be of importance, the organism must be present in large 
numbers. It is Gram-positive. It is seen without difficulty 
with high power, dry objectives in the fresh, unstained 
preparation. The bacilli are more frequently associated 
with malignant disease than with other conditions in the 
stomach. 

Trichomonas intestinalis 1 is rarely found in the stom- 
ach. Other protozoa — Balantidium coli, Lamblia intesti- 
nalis, Cercomonas hominis — are very uncommon. (For de- 
scriptions of these parasites see pp. 177-179.) 

Crystals are of little importance in the stomach. In 
bile-tinged specimens cholesterin crystals and spheres of 
leucin have been observed. Triple phosphate, fatty acid, 
and oxalic acid crystals have been noted. 

1 Colmheim, P. ' ' Inf usorien bei gut- und bosartigen Magenleiden nebst 
Bemerkungen ueber die sogenannte Inf usiorienenteritis. ' ' Deutsche med. 
Wchnschr., 1909, XXXV, 92. 



CHAPTER III 

THE FECES 1 

In the examination of the feces it is a matter of prime 
importance that the material be obtained as fresh as pos- 
sible. If the examination is delayed beyond a few hours 
at the most, it is quite possible, and. in fact, probable, that 
erroneous conclusions will be reached. This is particularly 
the case with regard to animal parasites. It is necessary, 
for example, to examine for ameba? before the material has 
cooled ; hookworm ova may hatch in the stool, under favor- 
able conditions, in twenty-four hours. Furthermore, bac- 
terial digestion of food rests, such as muscle fibers, may 
proceed to such a degree within a comparatively short time 
after the stool has been passed as to lead to a false impres- 
sion on microscopic inspection. Were it necessary, exam- 
ples might be multiplied almost indefinitely. 

MACROSCOPIC EXAMINATION OF THE FECES 

The approximate amount, form, consistence, and color 
of the stool are noted, and also all recognizable pathologi- 
cal elements, such as parasites, mucus, blood, pus, gall- 
stones, undigested portions of food, etc. A simple inspec- 
tion suffices for the determination of most of these points. 

1 As reference works in the study of the feces wide recognition has been 
accorded "Makro- und mikroskopische Diagnostik der menschlichen Exkre- 
niente" by M. L. Q. van Ledden Hulsebosch. Berlin, 1899, and to "Die 
Faezes des Menschen ' ' by Ad. Schmidt and J. Strasburger. Berlin, 1910, 3rd 
ed. Both are profusely illustrated. 

152 



THE FECES 153 

Amount.— The amount of the feces in health varies be- 
tween about 120 and 250 gm. in twenty-four hours. The 
frequency of defecation and the quantity of food eaten 
largely determine the amount passed at any one time. 

Form.— The formed or soft stool of the normal in- 
dividual requires no description. Scybali are the small, 
hard masses of fecal material which have remained in the 
bowel too long and have become abnormally dry. They 
are at times coated with mucus, and not infrequently fresh 
blood may be seen on their surface. The size of a formed 
stool should be noted; the small movements, about the 
thickness of a lead pencil, which are seen in starvation or 
in pathological states of the large intestine, are abnormal. 
In diarrhea the stools are fluid. 

Color.— The color of the stools in health is derived 
largely from (1) hydrobilirubin (urobilin), which is re- 
duced bilirubin; though in breast-fed infants' stools unal- 
tered bile pigment is met with. (2) Food may alter the 
color of the intestinal discharges. With a milk diet, the 
color is light. An unusually dark color results from eating 
blueberries, etc., and from drinking red wines. Vegetables 
rich in chlorophyll, such as spinach, may impart a dark 
green or olive tint. (3) Certain drugs have a marked effect 
on the color of the intestinal contents. After calomel a 
greenish color may be noted; bismuth salts may cause a 
dark brown or even a black color, due to the black crystals 
of bismuth suboxid. A normally pigmented stool, which 
becomes dark on exposure to the air, is often attributable 
to the use of iron. Similarly, if methylene blue be admin- 
istered by mouth, oxidation after the stool has been passed 
may lead to a dark bluish-green color on its surface. 

Among the abnormal coloring matters of the feces, (4) 
blood is of great importance. The extent to which the color 



154 MACROSCOPIC EXAMINATION 

is altered depends upon the size of the hemorrhage and its 
source — whether high or low in the gastrointestinal tract. 
Very small hemorrhages in the stomach or small intestine 
produce no perceptible change in the appearance of the 
feces; these are the so-called "occult hemorrhages," which 
are recognized only by chemical tests. On the other hand, 
large gastric or duodenal hemorrhages lead to the so-called 
"tarry" or black stools. A hemorrhage of any consider- 
able size low in the ileum often manifests itself by the pas- 
sage of very dark red clots or fluid, the hemoglobin show- 
ing less alteration than in the preceding instance. With 
rectal hemorrhages the blood is bright red, often unclot- 
ted, and is seen on the surface of the stool, not intimately 
mixed with it. (5) " Clay -colored" stools gain their name 
from the resemblance to white clay. They may be due to 
(a) entire absence of bile pigment, acholic stools, with the 
usual increase of fat which accompanies this condition; (b) 
the reduction of bilirubin by bacteria may be excessive, giv- 
ing rise to a colorless compound, leukohydrobilirubin. In 
this case the surface of the stool becomes dark after more 
or less prolonged exposure to the air, and, unlike acholic 
stools, hydrobilirubin is demonstrable; (c) with very exces- 
sive fat content, the normal fecal pigment may be so greatly 
obscured that the stool is clay-colored. (6) The presence 
of very large quantities of pus or of fluid may cover or 
dilute the normal pigment to such an extent that the speci- 
men appears lighter than usual. Pure pus, such as one 
sees after the rupture of an abscess into the rectum, needs 
no description. 

Mucus.— Mucus is present in normal feces, but not in 
sufficient quantity to be observed macroscopically. Long 
strings or ribbon-like masses of mucus, tenacious and slimy, 
usually slightly stained with urobilin, may be observed in 



THE FECES 155 

disease, or, again, the stools may be encased in mucus, giv- 
ing an appearance very suggestive of a membrane or a 
sausage-skin. More frequently smaller particles of mucus 
are found, varying in size from a split pea up to that of 
an almond or larger, at times blood-stained or mixed with 
pus or eosinophilic cells. 

Gall-stones.— Gall-stones or other concretions may be 
found, when present, in the following manner : A bowl or 
other vessel of about one liter capacity is lined with a 
double thickness of surgical gauze of sufficient size to per- 
mit the free margin to extend well beyond the edge of the 
bowl on all sides. The stool is now placed on the gauze 
in the bowl, and the free edges of the gauze are securely 
tied, so that the stool is contained in a bag of gauze. The 
specimen is left in the bowl, which is now placed under a 
stream of running water, where it is allowed to remain 
until all the finer particles of the feces have been washed 
away. Gall-stones, unless very minute, cannot pass through 
the meshes of the gauze, and are, therefore, found in the 
bag. 

This procedure is applicable to the detection of the lar- 
ger fruit seeds, foreign bodies, etc. 

Parasites.— Parasites, such as Ascaris lumbricoides and 
the larger cestodes, are striking objects which arrest atten- 
tion at once. Methods for the detection of the smaller 
worms are described in connection with the hookworm. 

INTESTINAL TEST DIET 

In the study of functional and anatomic alterations of 
the intestine, a uniform diet is desirable for many reasons ; 
microscopic and chemical examinations are greatly simpli- 
fied, and there is supplied a basis of comparison which is 



156 INTESTINAL TEST DIET 

not possible when patients are free to choose their own 
food. 

The test diet of Schmidt and Strasburger 1 is that gen- 
erally nsed. Five small meals are given, the first on wak- 
ing, the fourth in the afternoon. 

Diet No. I. — In the Morning. — One-half liter of milk, or 
of tea, or cocoa cooked with milk or water. One roll with 
butter and 1 soft-boiled egg. 

Breakfast. — One dish of oatmeal cooked with milk and 
strained, with salt or sugar as desired. Instead of oat- 
meal, gruel or porridge may be taken. 

Noon. — One-quarter pound of chopped, lean beef, 
broiled in butter, rare. A fairly liberal portion of potato 
puree. 

Afternoon. — Same as in the morning, without the egg. 

Evening. — One-half liter of milk or 1 dish of oatmeal 
prepared as for breakfast. One roll with butter. One or 2 
eggs, soft-boiled or scrambled. 

Diet No. II. — For quantitative studies Schmidt and 
Strasbnrger 2 recommend a diet containing the following 
foods, which must be carefully measured: 1.5 liters of 
milk, 100 gm. of zwieback, 2 eggs, 50 gm. of butter, 125 gm. 
of beef, 190 gm. of potatoes, oatmeal made from 80 gm. 
of dry meal, 2 to 3 gm. of salt. They suggest the follow- 
ing arrangement for giving the food : 

In the Morning. — 0.5 liter milk and 50 gm. zwieback. 

In the Forenoon.Str&med oatmeal prepared from 40 
gm. of oatmeal, 10 gm. of butter, 200 c. c. of milk, 300 c. c. 
of water, 1 egg, and salt. 

Noon.— 125 gm. of chormed beef (raw weight) broiled 

Schmidt, A., and Strasburger, J. Loc. cit., pp. 5-6. Also Schmidt, A. 
"The examination of the function of the intestines by means of the test diet, 
etc." (Translated by C. D. Aaron.) Philadelphia, 1909. 

2 Loc. cit. 



THE FECES 157 

with 20 gm. of butter; the beef should remain raw on the 
inside ; 250 gm. of potato puree prepared from 190 gm. of 
mashed potato, 100 c. c. of milk, 10 gm. of butter, and salt. 

Afternoon. — Same as in the morning. 

Evening. — Same as in the forenoon. 

The authors usually give the diet for three days, occa- 
sionally longer. To determine when the food has passed 
through the intestinal tract, they give 0.3 gm. of powdered 
carmine in capsule with the first meal of the test diet. The 
carmine produces a red color in the feces. 

This second diet, it is calculated, contains 102 gm. of 
protein, 111 gm. of fat, and 191 gm. of carbohydrate. It is 
equivalent to 2,234 calories. 

Weight of Dried Feces.— The feces are dried on a steam 
or water bath. The weight of the dried feces of normal 
adults on Schmidt's diet No. II varies between about 45 
and 62 gm. 



CHEMICAL EXAMINATION OF THE FECES 

For chemical examination a fresh specimen of feces 
should always be used. 

Reaction.— The reaction of normal feces is neutral, 
faintly alkaline, or faintly acid. It is tested with litmus 
paper. If the stool is formed or soft, a small portion for 
testing should be rubbed in a mortar with a little distilled 
water. 

Pigments.— The normal fecal pigment is hydrobilirubin 
(urobilin). In breast-fed children, however, the bilirubin 
is not reduced by bacteria, and appears as such in the feces. 
In disease unaltered bilirubin may be present in the feces ; 
the same is true after active purgation. 

12 



158 CHEMICAL EXAMINATION 

Ukobilin (Hydrobilirubin) 

(1) Schmidt's Test.— A portion of the fresh feces the 
size of a hazel-nut or larger is rubbed in a mortar witl] 
three to four times its volume of concentrated watery solu- 
tion of bichlorid of mercury. The suspension obtained is 
placed in a covered Petri dish, and set aside for twenty- 
four hours. All particles stained with hydrobilirubin (uro- 
bilin) are colored red, whereas bilirubin becomes green. 
The color change may appear in less than an hour. The 
material may be examined microscopically, when even mi- 
nute particles which contained bilirubin become evident 
by their green color. 

(2) Schlesinger's Test. 1 — A portion of the stool is 
rubbed in a mortar with distilled water to obtain a thin 
watery suspension. If much fat is present, extract the sus- 
pension with ether twice to remove it. Then treat the sus- 
pension with acid alcohol (HC1 3 c. c, alcohol to 100 c. c), 
and later neutralize the acid with ammonia. Now add to 
the mixture an equal volume of saturated alcoholic solution 
of zinc acetate, mix thoroughly, and filter. In the presence 
of urobilin a green fluorescence is seen. 

(3) Spectroscopic Determination.— The watery suspen- 
sion of feces is acidulated with acetic acid, and is then 
extracted with amyl alcohol. The extract is examined for 
the bands of urobilin (see p. 71). 

Bilirubin 

(1) Schmidt's Test.— This is applied as just described 
for urobilin. A green color denotes the presence of bili- 
rubin. It permits the recognition of even microscopic bili- 

1 Schlesinger, W. Loc. cit. (p. 71). 



THE FECES 159 

rubin-stained particles in the presence of an excess of 
urobilin. 

(2) Gmelin's Test.— This test is applicable only when 
a great excess of bilirubin is present (Schmidt and Stras- 
burger). The feces must be examined while fresh. A 
watery suspension of the material is prepared. Filter 
paper is soaked in the suspension, and then a drop of yellow 
nitric acid is placed on the paper. The characteristic play 
of colors is seen about the edge of the drop — yellow, red, 
violet, blue, and green, the last at the periphery. 

Blood 

(1) Weber's Test.— A watery suspension of feces is pre- 
pared in a mortar. If the stool contains much fat, this is 
removed by extraction with ether. Then add to the sus- 
pension one-third volume of glacial acetic acid and mix 
thoroughly. If blood is present, the coloring matter is 
converted into acid hematin. The mixture is now filtered 
and the nitrate extracted with two to three volumes of 
ether. Separation of the ether may be hastened by the 
addition of a few drops of alcohol. Depending upon the 
quantity of blood present, the ether extract shows a more 
or less intense shade of brown. The extract is now exam- 
ined spectroscopically for the bands of acid hematin (see 
p. 78). 

(2) The Guiac Test.— If the stool contains too little 
blood to give the spectroscopic test, about 2 c. c. of the 
ether extract obtained in Weber's test is treated with 
about 10 drops of freshly prepared tincture of guiac (a 
knife -point of powdered guiac dissolved in about 5 c. c. 
of alcohol), and 20 to 30 drops of hydrogen peroxid or 
old, ozonized turpentine. The mixture is shaken, and in 



160 CHEMICAL EXAMINATION 

the presence of blood a blue color develops throughout 
the mixture. The color fades after standing a few 
minutes. 

Cowie x reports a "water modification " which he finds 
more delicate. All glassware should be chemically clean 
and dry. One gram of feces which has been softened with 
as little water as possible is rubbed in a mortar with 4 to 
5 c. c. of glacial acetic acid. To the suspension obtained 
add 30 c. c. of ether, and shake. To 1 or 2 c. c. of the ether 
extract add an equal amount of distilled water, and shake 
thoroughly. Now a knife-point of powdered guiac is placed 
in the test tube, and is dissolved by agitating the contents. 
Finally 30 drops of old, water-white, pure turpentine (or 
hydrogen peroxid) are added, and the contents of the tube 
mixed. The tube is examined against a white background 
for the color reaction. If blood is present to the extent 
of 1 mg. in 1 gm. of feces, a distinct light blue color de- 
velops quickly in the ether. With larger amounts of blood 
the color is, of course, more intense. 

Sources of Error. — The guiac test is not a reliable test 
for blood. It is, however, a very delicate test, and, when it 
is negative, blood in appreciable quantity is absent. A 
positive reaction may be given by a great many substances. 
Of those most apt to lead to difficulty in fecal examinations 
raw meat, chlorophyll, pus, and salts of the heavy metals 
are familiar examples. It is advisable to exclude meat and 
green vegetables from the diet for at least three days be- 
fore collecting the specimen for examination. All drugs 
which might interfere with the test, such as preparations 
of iron, should also be discontinued. A full list of the sub- 

1 Cowie, D. M. " A comparative study of the occult blood tests ; a new 
modification of the guiac reaction; its value in legal medicine." Amer. Jour. 
Med. Sci., 1907, CXXXIII, 408. 



THE FECES 161 

stances reacting with guiac and similar substances — phe- 
nolphthalin, aloin, benzidin — is given by Kastle. 1 

The value of the test is concisely stated by Kastle, 1 who 
says : ' ' The general consensus of opinion among those 
who have given this subject their attention would seem to 
be that the guiacum test for blood and similar color reac- 
tions are valuable, especially if they lead to negative re- 
sults, as proving beyond the peradventure of a doubt that 
blood is absent. On the other hand, if a positive test is 
obtained, care should be taken to exclude oxidases or perox- 
idases by boiling, and the salts of the heavy metals and 
other oxidizing agents by chemical methods, and, if pos- 
sible, to subject the material under investigation to confirm- 
ative tests for blood before finally concluding that blood 
is present." 

(3) Teichmann's Hemin Crystal Test.— With a minute 
particle of dried feces, the hemin crystal test may be per- 
formed (see p. 82). With very small amounts of blood the 
test may fail. 

Fat and starch are usually recognized without difficulty 
by microscopic examination. For quantitative determina- 
tion of neutral fat and fatty acids, works on chemistry 
should be consulted. (A new method is described by Folin, 
0., and Wentworth, A. H. A new method for the deter- 
mination of fat and fatty acids in feces, Jour. Biol. Chem., 
1910, vii, 421.) 

Enzymes in the Feces 

The examination of the feces for enzymes of the pan- 
creas has received considerable attention. Trypsin and 
amylase (diastase) are most often determined, for their 
relations in the feces are best understood. 

1 Kastle, J. H. "Chemical tests for blood." Bull. No. 51, Hyg. Lab., 
U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1909, pp. 1-62. 



162 CHEMICAL EXAMINATION 

Trypsin 

Method of Gross 1 for the Determination of Trypsin.— 

Eeagents : 

Solution 1: 

Caseinum purissimum (Griibler). 0.5 gm. 

Sodium carbonate 1.0 gm. 

Distilled water 1,000.0 c. c. 

Dissolve by very gentle heating, if neces- 
sary. Add toluol to prevent bacterial growth. 

Solution 2: 

Sodium carbonate 1.0 gm. 

Distilled water 1,000.0 c. c. 

The feces to be examined are rubbed in a mortar with 
three times their bulk of solution 2, until a homogeneous 
suspension is obtained. This is filtered, till the filtrate is 
clear. Ordinarily the filtration causes no trouble, but if 
there is much turbidity from bacteria they settle to the 
bottom, and the clear fluid may be decanted (Gross). Ten 
c. c. of the fecal filtrate are placed in a flask with 100 c. c. 
of the casein solution (solution 1). A few c. c. of toluol 
are added to prevent bacterial decomposition of the casein. 
The flask is now placed in the incubator at 37° to 40° C. 
From time to time small portions are removed and tested 
for casein; this substance is precipitated by dilute (1 per 
cent.) acetic acid, though the products of its digestion are 
unaffected. The material is kept in the incubator till the 
casein has been completely digested. The time is noted. 

The rapidity of digestion (and the amount of trypsin) 



1 Gross, O. ' ' Znr Funktionspriifung des Pankreas. ' ' Deutsche med. 
Wchnschr., 1909, XXXV, 706. 



THE FECES 163 

varies with the diet. It is completed most quickly after 
a protein diet; with carbohydrate food the trypsin is dim- 
inished, while intermediate values are obtained with a diet 
largely of fat. The examination should be made after pro- 
tein diet; the average time required for complete diges- 
tion is 12 to 14 hours, the normal limits being 8 and 15 
hours. 

Amylase 

Wohlgemuth 's * Method for Determination of Amylase, 
as Modified by Hawk. 2 — "Weigh accurately about 2 gm. of 
fresh feces into a mortar (duplicate determinations should 
be made), add 8 c. c. of a phosphate-chlorid solution (0.1 
mol. dihydrogen sodium phosphate and 0.2 mol. disodium 
hydrogen phosphate per liter of 1 per cent, sodium chlo- 
rid), 2 c. c. at a time, rubbing the feces mixture to a homo- 
geneous consistency after each addition of the extraction 
medium. Permit the mixture to stand at room temperature 
for a half hour with frequent stirring. We now have a 
neutral fecal suspension. Transfer this to a graduated 15- 
c. c. centrifuge tube, being sure to wash the mortar and 
pestle carefully with the phosphate-chlorid solution, and 
add all washings to the suspension in the centrifuge tube. 
The suspension is now made up to the 15-c. c. mark with 
the phosphate-chlorid solution and centrifugated for a fif- 
teen-minute period, or longer, if necessary, to secure a sat- 
isfactory sedimentation. At this point read and record the 
height of the sediment column. Eemove the supernatant 

1 Wohlgemuth, J. (a) "Ueber eine neue Methocle zur quantitativen Be- 
stimmung des diastatischen Ferments." Biochem. Ztschr., 1908, IX, 1. (b) 
"Beitrag zur funktionellen Diagnostik des Pankreas. " Berlin. Idin. Wchnschr., 
1910, XL VII, 92. 

2 Hawk, P. B. "A modification of Wohlegmuth's method for the quantita- 
tive study of the activity of the pancreatic function." Arch. Int. Med., 1911, 
VIII, 552. 



lei CHEMICAL EXAMINATION 

liquid by means of a bent pipette, transfei it to a 50-:. 
volumetric flask, and dilute it to the 50-c. e. mark with the 
phosphate-chlorid solntion. Mix the fecal extract thor- 
oughly and determine its amylolytic activity. For this 
purpose a series : six graduated tubes is prepared, con- 
taining volumes of the extract ranging from 2.5 to 0.078 
c. c. Each of the intermediate tubes in this series will thus 
contain one-half as much fluid as the preceding tube. N w 
make the contents of each tube 2.5 c. c. by means of the 
phosphate-chlorid solution in order to secure a uniform 
electrolyte concentration. Introduce 5 c. c. of a 1 per cent. 
soluble starch solution and three drops of toluol into each 
tube, thoroughly mix the contents shaking, close the 

tubes by mean- : st - and place them in an incuba- 

tor " . for twenty-four hours. (In preparing the 1 

per cent, starch solution, the weighed starch powder should 
be dissolved in cold distilled water in a — le and 
stirre •.. until a homo2 suspension is obtained. The 

mixture should then be heated with constant stirring until 
it is clear. This ordinarily takes from eight to ten miu : 
A slightly opaque solution is thus obtained, which should 
be cooled and made up to the proper volume before using.) 
At the end of this time remove the tubes, fill each to within 
half an inch of the top with ice water, add one drop of 
tenth normal iodin solution, thoroughly mix the conten :s. 
and examine the tubes carefully with the aid of a strong 
light. Select the last tube in the series, which shows en: 
absence of blue color, thus indicating that the starch has 
been completely transformed into dextrin and sugar, and 
calculate the amylolytic activity on the basis of this dilu- 
tion. In case of indecision between fcwc tubes, add an extra 
drop of the iodin solution and observe them again. 

"The amylolytic activity, Df, of a given stool may 




THE FECES 165 

expressed in terms of 1 c. c. of sediment obtained by the 
centrifugation, as above described. For example, if it is 
found that 0.31 c. c. of the phosphate-chlorid extract of the 
stool acting at 38° C. for twenty-four hours completely 
transformed the starch in 5 c. c. of a 1 per cent, starch 
solution, then we would have the following proportion: 

0.31:5::1 (c. c. extract) :x 

The value of x in this case is 16.1, which means that 1 c. c. 
of the fecal extract possesses the power of completely di- 
gesting 16.1 c. c. of a 1 per cent, starch solution in twenty- 
four hours at 38° C. 

"Inasmuch as stools vary so greatly as to water con- 
tent, it is essential to an accurate comparison of stools 
that such comparison be made on the basis of the solid 
matter. Supposing, for example, that in the above deter- 
mination we had 6.2 c. c. of sediment. Since the super- 
natant fluid was removed and made up to 50 c. c. before 
testing its amylolytic value, it is evident that 1 c. c. of this 
sediment is equivalent to 8 c. c. extract. Therefore, in 
order to derive the amylolytic value of 1 c. c. of sediment, 
we must multiply the value (16.1) as obtained above for 
the extract by 8. This yields 128.8 and enables us to ex- 
press the activity as follows : 

Df HI = 128.8" 

This is the method of calculation employed by Wohlge- 
muth. The departure from the original technique, which 
Hawk has suggested, consists in the addition of the phos- 
phates to the chlorid solution. The object of this is to se- 
cure a uniform medium in which the amylase may be ex- 



166 MICROSCOPIC EXAMINATION 

anrined, for it has been shown that the reaction exerts a 
marked effect on its activity. To do away with this source 
of error the phosphate-chlorid mixture is employed. 

Wohlgemuth finds that the normal average value is 
about 150. 

MICROSCOPIC EXAMINATION OF THE FECES 

Unless the stool be very fluid, it is necessary to dilute 
it with water before examining it microscopically. The 
method proposed by Stiles is most satisfactory. A drop of 
water is placed upon a clean glass slide 1 and then, with a 
flat wooden toothpick or other suitable instrument, a small 
quantity of feces is transferred to the drop, and mixed 
with it. During the mixing the slide is inclined and the 
mixing is done with an upward stroke. By doing so all 
gritty, solid particles usually are deposited at the upper 
end, and do not interfere with the spreading of the speci- 
men under the cover glass, which is applied as soon as a 
thin, uniform suspension of the feces has been secured. 
The preparation is now ready for examination. Others 
prefer to examine the preparation without a cover glass. 
The specimen is of uneven thickness, and has the further 
disadvantage that parts of it become dry before the exam- 
ination can be finished. 

The specimen should be searched carefully with the low 
power, and doubtful objects should be examined with a 
dry lens of higher magnification. Among the objects to be 
seen in the microscopic examination are remnants of foods, 

1 Large slides, 2 by 3 in.., or small glass plates (photographic plates) are 
convenient for examining feces, as there is less danger of soiling the hands. 
The toothpick should, of course, be burned or placed in a disinfecting solution 
immediately after use. Feces should always be handled as infectious material, 
for it is impossible to know when one is dealing with a typhoid or other carrier. 



THE FECES 167 

bacteria, granular debris, cells from the mucosa, from 
blood, or from exudates, parasites, ova, and crystals. 

Food Eemnants 

Muscle fibers are always seen in the feces of patients on 
a mixed diet. They are yellow in color. As a result of 
digestion the ends of the fibers are usually rounded, and 
often only small particles remain. In some of the fibers, 
however, the striations are well preserved. When diges- 
tion of the fibers is faulty, their number is greatly in- 
creased, the striations are preserved in the majority, and 
the ends are square, not rounded. 

Fibrous connective tissue may be observed, particularly 
when there is a lack of free hydrochloric acid in the stom- 
ach, since the fibers, digestible in normal gastric juice, are 
unaffected by the intestinal and pancreatic secretions. 

Curds may be seen after a rich milk diet and are fre- 
quently encountered in infants ' stools. In the latter masses 
of fat — neutral fat and fatty acids — may bear a close re- 
semblance to curds on macroscopic examination. 

Vegetable cells are very varied in shape, 1 and at times 
are mistaken for parasitic ova by inexperienced workers. 
The cells are less regular in shape and not so uniform in 
size as parasitic ova. Measurements are not necessary to 
demonstrate the great variation in size; it is quite obvious 
from inspection alone. Often the addition of a drop of 
Lugol's solution to the specimen will stain the vegetable 
cells, or at least some of them, blue. If the starch has been 
digested, this reaction is lost. The larger sheets of vege- 
table cells cannot be mistaken for anything else. 

1 Hulsebosch (Joe. cit.) gives excellent illustrations of a great variety of 
vegetable cells. Schmidt and Strasburger also show many of the commoner 
cells. 



168 MICROSCOPIC EXAMINATION 

Vegetable spirals are the vessels of plants, which have 
escaped digestion in the intestine. When tightly coiled 
they present beaded borders with a latticed appearance be- 
tween; when drawn ont the spiral becomes evident. 

Vegetable hairs are not infrequently mistaken for the 
embryos of parasites. They differ from all known embryos 
parasitic in man in having a perfectly homogeneous wall, 
devoid of cellular structure, with a central canal extending 
throughout. Furthermore, unlike living embryos, they pos- 
sess no motility. They usually have a yellowish tint and 
are very refractive. 

Starch granules are infrequent in the stools. Their 
usually oval shape, laminated appearance, and the iodin 
reaction identify them. 

Unrecognizable debris is constantly seen in the stools. 

Fat in the stools is discussed on p. 171. 

Bactekia 

Bacteria of all forms are extremely numerous in the 
stools, except in the case of breast-fed infants. With a 
few exceptions, little diagnostic importance is placed in 
their study. In typhoid fever the simplicity and accuracy 
of blood cultures make fecal examination for Bacillus ty- 
phosus superfluous, excepting in the case of typhoid bacil- 
lus-carriers with gall-bladder infections. In bacillary dy- 
sentery a search for the Shiga bacillus may be desirable; 
the same is true of Asiatic cholera. (For methods of de- 
tecting organisms such as these in the feces, the reader is 
referred to works on bacteriology.) 

Tubercle bacilli are most readily found with the aid of 
the antiformin method (p. 214). It is necessary to cleanse 
the anus and surrounding parts in order to remove the 



THE FECES 169 

smegma bacilli, as they are normally present in this part 
of the body. Since tubercle bacilli are frequently present 
in the feces of patients with pulmonary tuberculosis, due 
to the habit many adults — and all very young children — 
have of swallowing the sputa, they do not necessarily in- 
dicate an intestinal lesion. It is only when the bacilli are 
demonstrated in mucopurulent or bloody masses that a 
diagnosis of intestinal tuberculosis is probable. (For the 
method of staining tubercle bacilli see p. 213.) 

Yeasts, often budding, may grow in the feces, and may 
be quite numerous. They may be present as a contamina- 
tion after the stool has been passed. 

Sarcince and Oppler-Boas bacilli may be conspicuous in 
the feces. Their occurrence in large numbers is probably 
always secondary to pathological conditions in the stom- 
ach favoring their growth. Gram's stain should be used to 
demonstrate the long bacilli, for other bacteria are so nu- 
merous that they are less evident in the fresh specimen 
than they are in the stomach contents. 



Cells 

Epithelial cells of the intestinal mucosa desquamate con- 
tinually. The single small, round, or oval nucleus is usu- 
ally visible in the cell ; if not, 3 per cent, acetic acid should 
be added. Normally, epithelial cells are few in number. 
They may be well preserved, but are frequently swollen 
or otherwise degenerated. They are often found imbedded 
in mucus. 

Blood is never found in normal intestinal contents. It 
is only when the hemorrhage occurs in the lower part of 
the intestinal tract that the morphology of the cells is suffi- 



170 MICROSCOPIC EXAMINATION 

ciently well preserved to permit recognition on microscopic 
examination. Shadows of the red cells may be seen. Clots 
often contain erythrocytes in a good state of preservation. 
For the chemical tests for blood see pp. 159-160. 

Pus cells — polynnclear neutrophilic leukocytes — in very 
small number, i. e., an occasional cell, are not patho- 
logical. If the cells are not degenerated beyond recog- 
nition, the distinguishing feature is the polymorphous 
nucleus, together with the finely granular protoplasm. 
The addition of dilute acetic acid may be necessary to 
demonstrate the nucleus. The cells may be free or em- 
bedded in mucus. 

Eosinophilic leukocytes are never found in normal feces. 
They are most frequently, though not always, 1 associated 
with intestinal parasites or protozoan infections. The cells 
are often found in particles of mucus, not infrequently 
blood-stained; Charcot-Leyden crystals are usually found 
among the eosinophiles. 

Ckystals 

Crystals - are commonly seen in the stools. Those of 
ammoniomagnesium phosphate are of frequent occurrence. 
They are often very imperfect. Crystals of calcium oxalate 
and of calcium phosphate are occasionally found. In addi- 
tion, calcium salts of unknown acids may be precipitated in 
the feces as irregularly round or oval, bile-stained masses, 
at times with concentric rings. Calcium soaps are con- 
stantly present. Cholesterin is rarely observed in crystal- 
line form. 

^angstein. L. "Zur Kenntnis eosinophiler Darmkrisen im Saugling- 
salter." Munchen. med. WcJimclir., 1911, LVIII, 623. 

2 The majority of these crystals have been described in the section on the 
urine, to which the reader is referred. 



THE FECES 171 

Fat. — Needle-like crystals of fatty acids and insoluble 
soaps are always encountered on microscopic examination 
of the feces. The needles are short and slender, and are 
generally massed, so that the outline of the separate crys- 
tals is more or less obscure. At times they are extremely 
abundant, so much so that they may form the bulk of the 
stool. Fatty acids are soluble in alcohol and ether; soaps 
are insoluble. Fatty acids are further differentiated from 
soaps by the fact that their crystals melt to form droplets 
on warming. Morphological differentiation between the 
two may be impossible. Soaps may be present in the form 
of scales or long needles arranged in clusters. Neutral fat, 
in the form of droplets, may be found in any normal stool. 
The nature of the droplets may be clear from their vary- 
ing size, high refractivity, and slightly greenish tint with 
strong illumination. To identify them with certainty, add 
to the specimen a drop of Sudan III or Scharlach E (satu- 
rated solution in 70 per cent, alcohol), by which the drop- 
lets of neutral fat are stained orange to deep orange-red, 
the intensity of the color depending largely on the size of 
the droplet. 

Charcot-Leyden crystals are never found in normal 
stools. They are always associated with the presence of 
eosinophilic leukocytes, though they may persist after 
the cells have disappeared. They are diamond-shaped, 
refractive bodies, which may be stained with eosin (see 
p. 211). 

Bismuth suboxid appears in the stools in crystalline 
form after the administration of bismuth salts by mouth. 
The crystals are black, irregular rhombs. They are fre- 
quently so abundant that the stool is dark or even black 
in color. 

Hematoidin crystals have been observed occasionally. 



172 MICROSCOPIC EXAMINATION 



Intestinal Pakasites 1 

In the Southern States and our island possessions in- 
testinal parasites of one sort or another are the rule rather 
than the exception. Indeed, among the poorer classes of 
the population practically all are infected in certain local- 
ities. The free communication between all parts of the 
country is constantly disseminating the parasites, so that 
they are becoming of greater general importance each year, 
A description is given, therefore, of the important para- 
sites, whose presence may be determined by fecal exam- 
ination. 

The low power objectives are used in the examination 
of the feces for protozoa, ova, and embryos, the dry objec- 
tives of higher magnification being employed for final iden- 
tification. 

Protozoa — Rhizopoda 

Entameba Histolytica.— Entameb a histolytica 2 (Fig. 
13C), found in the stools of those suffering with amebic 
dysentery, is a protozoan parasite belonging to the class 
Ehizopoda. When present in the stools, it is most readily 
found by selecting for examination particles of mucus, es- 
pecially those which are blood-stained. The mucus may be 
obtained from the stools after administering a saline ca- 
thartic, if necessary, or by passing the rectal tube and re- 

1 The laboratory worker is advised to consult the Bulletins of the U. S. 
Public Health and Marine Hospital Service, Washington. D. C. Those issuing 
from the Department of Zoology by Stiles and his co-workers are invaluable to 
physicians. The excellent book of M. Braun, ' ' The Animal Parasites of Man, ' ' 
translated into English by Falcke, Sambon, and Theobald, is also to be recom- 
mended to physicians interested in the parasites of man. 

2 For an excellent discussion of the amebse of man see Craig, C. F. tl The 
Parasitic Amoebae of Man." Philadelphia and London, 1911. 



THE FECES 



173 



moving the mucus which clings to the eye of the tube. In 
the absence of mucus, the fluid portion of the stool is ex- 
amined, after giving the patient a saline cathartic. Since 
the amebse usually become quiescent soon after the speci- 
men cools, it is absolutely essential that the material be 
examined at once, while still warm. A warm stage for the 
microscope is a great advantage, though not a necessity. 




Fig. 13. — Parasitic Amebj;. A, Entameba coli; B, Entameba tetragena; C, En- 
tameba histolytica; D, Diagrammatic; 1, Ectoplasm; 2, Endoplasm; 3, Nucleus, 
nuclear membrane, centriole; 4, Erythrocytes; 5, Vacuole. (Adapted from 
Craig.) 



In winter, placing the microscope on a radiator often fur- 
nishes enough heat to keep the parasites actively motile. 
Entameba histolytica measures 0.010 to 0.035 to 0.070 
mm. in diameter, though young forms which are smaller 
may be found. The majority of those seen in the stools 
are between 15 and 45 micra. The ameboid parasite pos- 
sesses ectosarc and endosarc, which are well differen- 
tiated. The ectosarc has a peculiar greenish color, and, 
when thrown out to form a pseudopod, it presents an ap- 
pearance suggesting that of ground glass. It is highly re- 

13 



174 MICROSCOPIC EXAMINATION 

fractive. The endosarc or endoplasm is granular, and con- 
tains one to ten or more vacuoles — the younger forms usu 
ally only one — which are not contractile. If the intestinal 
lesions have been bleeding, many engulfed red corpuscles, 
often in a fair state of preservation, are seen in the endo- 
plasm. The nucleus is not seen, as a rule. The parasite 
is possessed of ameboid motion which, in a fresh, warm 
specimen, is very active, rapid, and progressive, the para- 
site changing its position in a few seconds, so that it may 
cross the field of the microscope. The ectoplasm is first 
protruded to form a pseudopod, and then the endoplasm 
flows into it. A whirling or circular motion of the endo- 
plasm is not infrequently observed. When more sluggish, 
there may be simply protrusion and retraction of pseudo- 
podia without change of position of the parasite. 

The motility of the parasite is necessary for its recog- 
nition. In fact, it should be a rule in the diagnosis of 
amebic infections, to which no exceptions should be made, 
to refrain from calling any cell an ameba unless actual 
ameboid motion has been observed in an otherwise charac- 
teristic organism. 

Simon 1 recommends staining the fresh specimen with 
dilute neutral red. A drop of dilute aqueous solution of 
the stain is allowed to run under the cover glass, or a mi- 
nute particle of the powdered stain may be added to the 
specimen. There is a selective staining of the endoplasm 
of the parasite. Ameboid movements seem not to be in- 
terfered with. The organisms stand out very prominently. 

Stained Preparations. — It is difficult to obtain satisfac- 
tory stained specimens of amebae. They may be stained 
with hematoxylin or with one of the Eomanowsky stains. 

1 Simon, C. E. " Clinical Diagnosis. ' ' 7th Ed., p. 214, 1911. Philadelphia 
and New York. 



THE FECES 175 

Brem 1 has had excellent results with a technique of his 
own. Cover slip preparations are made from the bloody 
mucus, and the specimens are then stained with Wright's, 
Hasting 's, Leishman's, or Wilson's stain in the following 
manner : 

(1) The unfixed specimen is covered with four drops 
of the stain, which is allowed to act for 10 to 15 seconds. 
Since the stain is dissolved in absolute methyl alcohol, this 
fixes the specimen. 

(2) Add to the stain four drops of distilled water. At 
the end of one minute — 

(3) Add four more drops of stain. Again, at the ex- 
piration of one minute — 

(4) Add four drops of water. The specimen is thus 
covered with a mixture of stain and water in equal quan- 
tities. This is permitted to act for 10 to 30 minutes. 

(5) The specimen is now washed in distilled water. 
(The cover glass should be kept level, while a stream of 
water is directed against its surface. In this way the pre- 
cipitated stain is washed or floated off ; dumping the stain- 
ing mixture from the specimen causes the precipitate to 
adhere to it.) The specimen is quickly dried by holding it 
over a small flame or by blotting carefully, and is mounted 
in balsam. 

The ectosarc of the amebse is stained dark blue, the 
endosarc a light blue. The nucleus takes a brilliant pur- 
plish-red color, and bacteria contained in the endosarc have 
a somewhat similar color. Phagocyted erythrocytes show 
a pinkish tint. 

Entameba Tetragena.— Entameba tetragena (Fig. 13B), 
which is also a cause of amebic dysentery, resembles both 

1 The method, devised by Dr. Walter Brem of Los Angeles, is unpublished 
and is given here with his kind permission. 



176 MICROSCOPIC EXAMINATION 

Entaineba histolytica and the non-pathogenic Entameba 
coli. Its diameter varies between 10 and 50 niicra, the ma- 
jority of the parasites being about the size of Entameba 
histolytica. Like the latter, it has well differentiated ecto- 
plasm and endoplasm. Xon-contractile vacuoles are ob- 
served in the endoplasm and also erythrocytes, when they 
are present in the feces. The nucleus of the parasite usu- 
ally may be seen very distinctly in the endoplasm — a point 
of resemblance to Entameba coli. The motility of the 
parasite is quite like that of Entameba histolytica. The 
organism is sail to stain poorly with Wright's stain 
(Craig). Apparently better results have been obtained 
with hematoxylin and eosin and other stains (Wetmore). 

Entameba Coli.— Entameba coli (Fig. 13A), the non- 
pathogenic ameba of man, may be found in the feces of 2 
to 6d per cent, of healthy individuals after the administra- 
tion of saline purgative (Craig). It differs from the 
pathogenic ameba? in (a) its smaller size, the majority of 
the parasites measuring 10 to 30 micra in diameter; (h) 
lack of sharp definition between ectosarc and endosarc; 
(c) presence of an easily recognizable nucle;:-. as in tetra- 
gena; (d) its opaque grayish color, especially well seen in 
the younger form- . the small number of vacuoles an I 

absence of erythrocytes in the endosarc (rarely a few rel 
corpuscles may be engulfed when they are present in th^ 
feces) ; (f ) the very sluggish ameboid movements with 
little, if any, change in position in the specimen; and, fi- 
nally, (g) its reaction to the Eomanowsky stains, the ecto- 
sarc taking a light blue, the endosarc a dark blue, color — 
just the reverse of the conditions seen in histolytica. 

Eesistant encysted forms, by which the infection is 
transmitted, have been described for the three species of 
ameba? considered above. 



THE FECES 177 

Flagellata 

Cercomonas Hominis.— Cercomonas hominis, a flagel- 
late, is, like Trichomonas intestinalis, probably non-patho- 
genic for man. It is a pear-shaped organism, pointed pos- 
teriorly, and measures 0.010 to 0.012 mm. in its long axis 
(Braun). At the anterior, rounded end a single whip or 
flagellum about half this length is attached. A nucleus is 
distinguishable at times near the anterior end. The para- 
site is actively motile, being propelled by its flagellum. 
Two or more parasites may become attached to one another 
by their posterior, pointed ends. The fluid portion of the 
stool should be examined. The specimen must be examined 
while perfectly fresh, as the organism dies quickly or 
assumes a spherical form. 

Trichomonas Intestinalis.— Trichomonas intestinalis 1 
(Fig. 14) is a pear-shaped body measuring 0.010 to 0.015 
mm. long and 0.003 to 0.004 mm. wide 
(Braun). Freund's measurements are 
somewhat greater. His smallest organ- 
ism was 0.009 mm. long and 0.0065 mm. 
wide, the largest 0.032 mm. long and 
0.019 mm. wide. The average of his 
measurements placed the length at 0.017 
mm., the width at 0.010 mm. The pos- 

. , _ ' . Fig. 14. — Trichomonas 

tenor end or the parasite is pointed. intestinalis. xabout 
Four flagella are attached to 'the an- 120 °- (Af ter Freund ° 
terior, rounded end, and there is an undulating membrane 
running from the point of insertion of the flagella to the 
posterior extremity of the organism. The body of the 
parasite is quite refractive and has a greenish, glass-like 

1 Fremiti, H. "Trichomonas hominis intestinalis; a study of its biology 
and its pathogenicity." Arch. Int. Med., 1908, I, 28. 




178 



MICROSCOPIC EXAMINATION 



appearance in a strong light. Its motility is often so ex- 
treme that many of the details of structure of the parasite 
are seen with difficulty, if at all. The fluid portion of a 
perfectly fresh stool should be taken for examination. 
Trichomonas intestinalis is probably non-pathogenic. En- 
cysted forms occur. 

Lamblia Intestinalis.— Lamblia intestinalis (Fig. 15), a 
third non-pathogenic flagellate parasitic in man. appears 
to be of less frequent occurrence than the two preceding. 
Viewed ventrally or dorsally, the parasite is pear-shaped. 





m 




Fig. 15. — Lamblia entbsixkaijb. Ventral and lateral views: on the intestinal 
epithelial cells; and encysted. (After Grassi and Schewiakoff, from Braun.) 



It is provided with a cup-like depression or excavation near 
its anterior extremity, by means of which it attaches itself 
to the epithelial cells of the mucosa of the small gut. The 
length of the parasite varies between 0.010 and 0.021 mm., 
the breadth between 0.005 and 0.012 mm. It is provided 
with eight flagella 0.009 to 0.014 mm. long, arranged in 
pairs. The first pair arises from the anterior, the second 
and third pairs from the posterior, end of the cup-like de- 
pression, while the fourth pair is attached to the posterior, 
pointed extremity of the body. The protoplasm is finely 
granular. The nucleus may be seen at times beneath the 



THE FECES 



179 



depression. If intestinal peristalsis is normal or dimin- 
ished, the forms just described may not be observed in the 
stools. In their stead encysted forms, which are not char- 
acteristic morphologically in the fresh specimen, are evac- 
uated with the feces. Acceleration of peristalsis from 
whatever cause may lead to the presence of the active stage 
of the parasite in the stools. 



Infusoria 

Balantidium Coli.— Balantidium coli (Paramecium coli) * 
is a protozoon which may be pathogenic for man, pro- 
ducing a disease somewhat analogous c 
to amebic dysentery with ulcers in the 
colon. It is very common in hogs. 
The parasite (Fig. 16) is oval, 0.060 
to 0.100 mm. in its long diameter by 
0.050 to 0.070 mm. The anterior end 
is less pointed than the posterior. A 
funnel-shaped peristome is situated 
anteriorly, about which are numerous 
cilia. Cilia are also conspicuous on the 
surface of the parasite. Ectosarc and 
endosarc are visible. The latter is 
granular and may contain fat droplets, 
bacteria, mucus, at times erythrocytes and pus corpuscles. 
Two or more contractile vacuoles are found in the endo- 
sarc. A macronucleus, rather kidney-shaped, and a round 
micronucleus, situated posteriorly, may be seen. Encysted 
forms, by means of which the infection is transferred, are 
described. 




Fig. 16. — Balantidium 
coli : a, nucleus ; b, va- 
cuole; c, peristome; 
d, food mass. (After 
Leuckart.fromBraun.) 



1 Bowman, F. B. ' ' The pathogenesis of Balantidium coli. 
A. M. A., 1911, LVII, 1814. 



Jour. 



181 MICROSCOPIC HXAM I NATION 

Dipterous Larvae. — Larva? of dipterous insects 1 (flies) 
appea r in tlie feces myi : - i - Hiey are easily ree- 

-i :- 7"...- : izi^T i. :~liz "::"":: :n - _ : ; 1 :_ ;"_ in 
length, according to the species, broad at one end, tapering 
at the other, and nsnally be - el ~ ilk little spines or hairs i - 
sufficiently diagn >sti lanson 2 ). It is particularly im- 

rorrazi: :i:;- :L-e ^:e:-iiiri. : : -: :.: : •_:. : l . :: :_ ' - ::— _ ::: 
~:~: :v.- rea= :>ns. 

and worms, constitate a large clas- 

nnmber of which are parasitic in man. Their anatomy and 

biology, thongh of _ :. are tonched upon in the 

foil wing pages nly in so far are of diagnostic 

: \ 

Necator Americanns.— Xecator americanns (Uncinaria 

Id hookworm, was first described 

by Si in 1902 7 .ether with its cousin of the Old 

rid, it is by far the most important nematode parasitic 

in man in ~ It > . :: "- :. --'. : : r 

isea oncinai : : anchylostomia - 

Diagnosis of infection with Xecator americanns is made 
by examination of the stools for the ova of the parasite. 
The gf ecimen of feces for examination should be fresh; in 



1 Gilbert. :" Infection off mam hy ffipterwas lame, viflL report off fewer 

3908, II, 226L (UbaatmH.) 

: :iizi:z 7 T: ■ t : : i.". I .h:^= - -\ :z '-? I z '\ 

m W A aew species off hookworm {TJiaenBaria anerkana) 

parasitie in mar. A - IfeA, J9©2, HL, 523. (b) "Tie sagadifieuiee off Ike 

recent American eases off bookworm disease (lEndanariasis or ane%lo£8oniasis) 
im majL" Bepriait, IStk asm. repu, Braeam Amm. Is Insfrj 1901). Wask, 

1 . EfT :- :. -i- rrfTLri?: '■ I ,- .::;.' ?rr: r: r : i :•:- 

worm dis-: : : : " - • ; i : - -"----.: 5 

; : H72 Li: T c ~ :": Ers'.:i i- Hi: " - - :~ ~~?i 1 : " it 
1-122 







THE FECES 181 

older specimens — 24 to 48 hours — the eggs may have 
hatched, in which case the embryos, much like those of 
Strongyloides stercoralis, may be observed. The specimen 
is examined with low power objective. All doubtful objects 
are examined with higher magnification. The ovum of the 
hookworm is characteristic, and, when once seen, can scarce- 
ly be mistaken for other bodies in the stool. 

The ova (Fig. 17) of Necator 
americanus are oval and possess a 
clear, colorless shell, which meas- 
ures 0.064 to 0.076 mm. by 0.036 to 
0.040 mm. (Stiles). The outline 
of the egg is sharp and clearly de- 
fined. Inside the shell is the yolk, Fig. 17. — Ovum of necator 

„v- i i. T 1 1 AMERICANUS. X460. 

which is unsegmented when de- 
posited by the female in the intestines, but usually presents 
two, four, or eight segments or cells, sometimes more, by 
the time it is evacuated with the feces. The yolk is dark 
gray or brownish-gray and finely granular; usually a 
lighter area representing the nucleus may be observed near 
the center of the yolk cells, especially when their number 
is' eight or less ; as the cells multiply, the decrease in size 
makes this area less conspicuous. 

In examining a preparation for hookworm (or other) 
ova, a mechanical stage is a great convenience. About one- 
half the area of the usual 3xl-in. slide should be covered 
with the diluted feces, and, before rendering a negative 
diagnosis, ten such specimens should be examined (Stiles). 
" ... To stop after finding a few hookworm eggs is 
not good practice. The examination should be continued 
to find, if present, eggs of other parasites, which are likely 
to be present in small numbers, and to get some idea of 
the number. When less than ten female worms are pres- 



182 MICROSCOPIC EXAMINATION 

ent, there may be an average of less than one egg to a 
slide" (Dock and Bass 1 ). 

Special Methods. 2 — When the eggs are very few. great- 
er diagnostic accuracy is attained by resorting to special 
methods for their detection. 

(1) Pepper's Method. 3 — Pepper has found that hook- 
worm ova possess a peculiar property of sticking to 
glass. If the preparation for microscopic examination 
be allowed to settle, immersion in water will remove the 
greater part of the fecal matter, while the hookworm 
ova stick to the slide. Dock and Bass find that bet- 
ter results are obtained with the method if part of 
the fecal material is first removed by centrifugaliza- 
tion. 

(2) Stiles' Method of Washing and Sedimenting. 4 — 
"Take one to two ounces of feces, mix with water, and 
place in a large bottle, retort, jar, or any other receptacle; 
add enough water to make from a pint to two quarts, ac- 
cording to the amount of feces; shake or stir thoroughly 
and allow to settle ; pour off the floating matter and the 
water down to near the sediment; repeat the washing and 
settling several times,' or as long as any matter will float. 
The last time this is done use a bottle or graduate with a 
smaller diameter, and, when the material is thoroughly 
settled, examine the fine sediment. It will be found that 

1 Dock, G., and Bass, C. C. "Hookworm Disease. Etiology, pathology, 
diagnosis, prognosis, prophylaxis, and treatment." St. Louis, 1910. (An ex- 
cellent discussion of hookworm disease in all its medical aspects, to which the 
reader is referred.) 

2 Hall, M. C. "A comparative study of methods of examining feces for 
evidences of parasitism." Bull. no. 135, Bureau Anim. Indust., U. S. Dept. of 
Agric, Wash., 1911. 

3 Pepper, Wm. ' ' A new method of examination of the feces for the ova of 
uncinaria. " Jour. Med. 'Research, 1908, XIII, 75. 

4 Loc. cit. (c), p. 85. 




THE FECES 183 

the eggs have settled more numerously in the fine sedi- 
ment than in the coarse material." 

(3) Centrif legalization. — Simple centrifugalization of 
the diluted feces often gives disappointing results. The es- 
sentials of the method, as given by Dock and Bass, 1 follow: 
"The feces should be diluted and well mixed with ten or 
more times their bulk of water. This should be strained 
through two or three layers of gauze in a funnel to remove 
the coarse particles. The exact length of time necessary 
to centrifuge, in order to throw most of the eggs suspended 
in water to the bottom of the tube, should be determined 
by experimenting with a known specimen that has already 
been washed once or twice and contains many eggs. This 
must be determined with the particular centrifuge used. 
. . . As the first diluted feces are much thicker than the 
washed feces and eggs on which the working time of the 
centrifuge has been determined, the eggs will go down 
somewhat slower the first time. It is, therefore, a good 
plan to centrifuge double time at first. If, for example, 
the working time of the centrifuge is four seconds, the first 
centrifuging should be eight seconds. This throws to the 
bottom most things heavier than eggs — like crystals, sand, 
large vegetable cells, etc. — and all eggs present. There 
remain suspended in the supernatant fluid nearly all bac- 
teria and fine particles, and many coarse particles lighter 
and more irregularly shaped than eggs. If the centrifuge 
is run longer, many of these go down, which is, of course, 
undesirable. Pour off this fluid and two-thirds, and often 
more, of the feces are removed by this washing. Refill the 
tube to about three-fourths its capacity, shake up thor- 
oughly, and centrifuge again, running now only the work- 
ing time of the centrifuge. It is important not to centri- 

1 Loc. tit., pp. 172 et seq. 



184 MICROSCOPIC EXAMINATION 

fuge longer than the working time of the centrifuge, as 
many fine and light particles would otherwise be thrown 
down. Considerable material remains suspended and may 
be removed by pouring ofT the supernatant fluid. Again 
the tube is filled, shaken, and centrifuged a proper length 
of time, and generally this will be sufficient for practical 
purposes. A part or all of the sediment is removed with 
a pipette, spread out on a slide, and examined for eggs. 
It consists of crystals, sand, and heavy, coarse food par- 
ticles, and eggs, if present. . . . Great care must be 
exercised to clean the centrifuge tubes before using them 
after they have had eggs in them. A proper centrifuge 
brush is serviceable. The method . . . permits the 
finding of eggs when less than half a dozen laying females 
are present, and often when only one is present. It is of 
additional service because it permits at the same time diag- 
nosis of infections with many other worms by which fewer 
eggs are laid, such as the tenias, oxyuris, bothriocephalus, 
etc." 

The adult worms of Necator americanus (Fig. 18) are 
found in the stools only after treatment. They are small, 
whitish, grayish, or reddish-brown in color, and have the 
anterior end curved dorsally to form a hook. The males 
measure 6 to 10 mm. in length, the females 8 to 15 mm. 
The stools, collected after treatment, are placed in a bucket 
or other suitable receptacle, stirred with several times their 
volume of water, and allowed to settle a few minutes, when 
the supernatant fluid is poured off. The washing is re- 
peated several times, and, finally, the sediment is trans- 
ferred to a plate, preferably Avith a black background, and 
the worms looked for. They may be identified by exam- 
ination under the microscope. 

Ankylostoma Duodenale.— Ankylostoma duodenale, the 



THE FECES 



185 



Old World hookworm, is of frequent occurrence in this 
country, often associated with Necator americanus. The 
methods of diagnosis are those described for Necator amer- 
icanus. The ova (Fig. 17) are identical in appearance. 




Fig. 18. — Necator americanus. Upper half males, lower half females. Inch 
measure. (After Dock and Bass.) 



Measurements show, however, that they are a little smaller 
than those of the New World variety, measuring 0.052 to 
0.061 by 0.032 to 0.038 mm. The differences are so slight 
that simple microscopic inspection does not suffice for the 
separation of the two. The adult parasites are a trifle lar- 
ger than Necator americanus. The males are 8 to 11 mm. 
long and 0.45 mm. wide; the females- 10 to 18 mm. long 



186 



MICROSCOPIC EXAMINATION 



and 0.6 mm. wide. There are certain well marked pecu- 
liarities by which the two species of hookworm may be 
differentiated. 

Strongyloides Stercoralis.— Strongyloides stercoralis 
(Strongyloides intestinalis), the parasite of Cochin China 
diarrhea, first reported in this country by Strong x and 
Thayer, 2 has proved to be widely spread through the South- 




Fig. 19. — The Rhabditiform Embryo of Stroxgyloides stercoralis. X460. 



ern States, as Thayer predicted. Diagnosis of infection 
is made by the finding of the actively motile rhabditiform 
embryos in the stool. 

Perfectly fresh feces should always be used for exam- 
ination. If the specimen is kept too long, the embryos 
may die or may change into the filariform larva?. If the 
stool is formed, fluid about it may contain the embryos, or 
they may be looked for in a preparation of the diluted 
feces. Since this often fails, even with heavy infections, 

1 Strong, E. P. "Cases of infection with Strongyloides intestinalis (first 
reported occurrence in Xorth America)." Johns Hopkins Hosp. JRep., 1902, 
X, 91. 

2 Thayer, W. S. "On the occurrence of Strongyloides intestinalis in the 
United States." Jour. Exp. Med., 1901, VI, 75. 



THE FECES 



187 



it is better practice to give a saline cathartic, if necessary, 
and examine the fluid stools. The embryos (Fig. 19) are 
0.450 to 0.600 mm. long and 0.016 to 0.020 mm. thick 
(Blanchard), and have a characteristic wriggling or 
squirming motion in the fresh stool. They are grayish- 
white and quite refractive. They possess a rhabditiform 
or bottle-shaped esophagus. Embryos which have died 
are, of course, less conspicuous, but, if well 
preserved, are characteristic. If the in- 
fected stool be kept for one to two days un- 
der suitable conditions of light, moisture, 
temperature, and oxygen supply, the rhab- 
ditiform embryos may develop into the 
filariform larvae, the infecting stage of the 
parasite. 

Ova of Strongyloides stercoralis (Fig. 
20) are extremely rare in the feces. They 
resemble the ova of the hookworm. In one 
of his cases Thayer found two eggs on daily 
examination of the stools for several months. 

Hookworm embryos 
which have developed 
in the stools (twenty- 
four to forty-eight 
hours after passage) 
may be mistaken for 
the embryos of 
Strongyloides sterco- 




Fig. 20. — Ovum of 
Strongyloides 
stercoralis . 
(DrawnwithLeitz 
obj. no. 7, ocular 
no. 3.) (After 
Thayer.) 





the former 
occur in the 



Fig. 21. — The Rhabditiform Embryo of Strongy- ra |' . 

LOIDES STERCORALIS (1) AND THE EMBRYO OF ' 

the Hookworm (2). Showing the difference in never 
length of the buccal capsule. Diagrammatic. 

freshly evacuated 
feces. They are most readily differentiated by the fact that 
the buccal capsule (Fig. 21) is very short in the embryo of 



188 MICROSCOPIC EXAMINATION 

Strongyloides stercoralis, relatively long in the hookworm 
embryo (Stiles). In case of doubt, a perfectly fresh stool 
should be obtained after a saline cathartic, when ova with- 
out embryos are found with hookworm infection alone ; with 
double infection, both embryos and ova are seen. 

Students often confuse plant or vegetable hairs with 
(dead or inactive) embryos. The former are distinguished 
by a straight central canal with hyalin, refractive walls of 
quite uniform thickness and devoid of finer structure. 

The adult parasite, which inhabits the small intestine, is 
probably a parthenogenetic female, and is a rarity in the 
stool. It is quite minute — 2.2 mm. long and about 0.034 mm. 
thick. 

Oxyuris Vermicularis.— Oxyuris vermicularis {Ascaris 
vermicularis), the common pinworm or seatworm, is a small 
nematode, the males measuring 3 to 5 mm. long with the 
posterior end curved, the females about 10 mm. long and 
0.6 mm. thick. The ova (Fig. 22) are flattened on one side, 
measure 0.050 by 0.016 to 0.20 mm. (Braun), 
and have a clear, thin shell. The ova are de- 
posited with the embryos already developed 
within the shell. It is necessary to recall the 
fact that the gravid females habitually wander 
from the region of the cecum and appendix to 
the rectum, anus, perineum, etc., in order to 
Fig. 22.— Ovum appreciate clearlv the reasons for the methods 

of Oxyuris 

vermicularis. of diagnosing infection. The crawling of the 
worms over the skin causes itching, so that 
the patient usually scratches the affected part. 

The presence of pinworrns may be determined 1 by (1) 
examining the feces for the adult worms (p. 184). Usually 
only females are found. The material for examination 

utiles, C. W. "Osier's Modern Medicine," Vol. I, 1907, p. 601. 




THE FECES 



189 



is best obtained by an enema given in the evening. (2) 
The worms may be seen in the crotch, especially if the 
child be examined during the restless period after retiring. 
(3) Microscopic examination of the scrapings of the skin 
about the anus or of dirt from the finger nails (the ova 
being picked up in scratching the perineum) may reveal 
the ova. (4) Eggs may be found in the feces. Fecal exam- 
ination for the eggs of the parasite is the least trustworthy 
of the methods of diagnosing infection. Stiles' statement, 
which is agreed to by experienced observers, should be 
borne in mind. He says: "The writer's experience is that 
the eggs may be found in fecal examination in some cases 
in which pinworm is not even suspected; but that a nega- 
tive examination is not of much value. ' ' Examination for 
the mature worms or for the ova in the scrapings of the 
perineum or finger nails give more reliable results. 

Trichuris Trichiura.— Trichuris trichiura (Trichoce- 
phalus dispar), the whipworm, is rarely seen in the feces. 
It may be found after treatment has been 
administered. The males are 35 to 45 mm. 
long, the females 35 to 50 mm., three-fifths 
of which is formed by the anterior filamen- 
tous portion (Blanchard). Diagnosis of in- 
fection is made by finding the ova in the 
feces. The eggs (Fig. 23) are oval in shape, 
with a relatively thick shell, which is gener- 
ally stained dark yellowish-brown. At either 
pole there is a space in the shell, which is 
occluded by a plug, the outer surface of which 
projects slightly beyond the shell. Within the shell the yel- 
lowish or brownish granular yolk substance is seen. The 
dimensions of the eggs are 0.050 to 0.056 mm. long and 
0.024 mm. wide (Blanchard). Though smaller than many 

14 




Fig. 23. — Ovum 
of Trichuris 
t r i ch IURA. 
X460. 



190 



MICROSCOPIC EXAMINATION 



other eggs, they are. nevertheless, of sufficient size to be 
easily seen with the usual low magnifications. 

Ascaris Lumbricoides.— Ascaris lurabricoides, the or- 
dinary "roundworm" of man, is the largest of the com- 
moner parasitic nematodes. Diagnosis may be made by 
the discovery of the parasite in the feces or vomitns. or of 
its ova in the stool. The living worm has a reddish or 
grayish-yellow color. The males vary in length between 





Fig. 24. — Ovum of Ascaris lumbricoides (1) ; the Same Under High Focus, Show- 
ing the Albuminous Coating (2). X-460. 



15 and 25 cm., and are about 3 mm. thick. The posterior 
end is conical, and is curved ventrally. Females are 20 
to 40 cm. long and about 5 mm. in thickness. Lateral, dor- 
sal, and ventral stripes rim longitudinally along the body 
of the parasite, the first being the most prominent. The 
ova (Fig. 24, 1 and 2) are elliptical and have a thick, trans- 
parent shell, which at times appears laminated. A rough 
albuminous coating forms the outer surface of the egg, and 
is usually stained brown with the fecal pigments. The al- 
buminous coating may be lost in some of the eggs. The 
size of the ova varies between 0.040 to 0.050 by 0.050 to 



THE FECES 



191 



0.070 mm. (Braun). Unfertilized ova 1 (Fig. 25) may be 
encountered. They are flatter — much less plump than the 
fertilized specimens — the shell is thinner, and the albumi- 
nous coating appears to be less abundant. The yolk is 
coarsely granular, in contrast to the finely granular ap- 
pearance in the fertilized eggs. 

Toxocara Canis.— Toxocara canis (Ascaris mystax), the 
common roundworm of dogs and cats, is rare in man. 2 The 

adult parasite is much smaller than As- 

caris lumbricoides ; the males are 4 to 6 
cm. long and 1 mm. thick, while the fe 
males measure 6 to 11 cm. in length and 
1.7 mm. in thickness. The maximal length 
recorded is 20 cm. (Blanchard). The ova 
resemble those of Ascaris lumbricoides, 
but are more spherical, having a diameter 
of 0.068 to 0.072 mm. (Blanchard). 

Trichinella spiralis.— The adult males 
of Trichinella spiralis are 1.4 to 1.6 mm. 



y^T* . 




J^BeS^n* \ 








SvU 


/ 


l ^$S§& 


] 






1 


§111111 




( 




w\ ) 


pH^^P 




^ng 





long and 0.04 mm. thick ; the females FlG - 25.— Unfertilized 

Ovum of Ascaris 

3 to 4 mm. in lumbricoides. 

X460. 

■ 1 :i :■ ' 



are larger, measuring 6 to 4 mm. in 
length, with an average thickness of 
0.06 mm. (Blanchard). They inhabit the small intestine. 
The feces, obtained by active purgation, are mixed thor- 
oughly with water, placed in a tall cylinder, and after the 
sediment has settled the fluid is poured off. The sediment 
is then placed in a dish with dark background; the thick- 
ness of the fecal layer should not exceed 1/12 inch. The 
dish is tilted, and any minute, hair-like objects are trans- 
ferred to a slide and examined microscopically (Stiles). 

1 Logan, O. T. " The little known atypical (unfertilized) egg of Ascaris 
lumbricoides." N. Y. Med. Jour., 1907, LXXXVI, 1164. 

2 Biesele. ' ' Ueber einen Fall von Ascaris mystax beim Menschen. ' ' 
Miinchen. med. Wclinschr., 1911, LVIII, 2391. 



192 MICROSCOPIC EXAMINATION 

The females may remain in the intestines eight weeks ; the 
males die within a few clays. The embryos may be re- 
covered from the blood (p. 310). 

Trematodes 

Trematodes * or flukeworms are fortunately rare in the 
United States, the cases reported being largely importa- 
tions from Asia and Africa. Of those whose presence in 
the body may be determined by finding the ova in the feces, 
the following are among the more important. The para- 
sites themselves are rarely seen during the life of the host. 

Opisthorchis Sinensis.— Opisthorchis sinensis, a liver- 
fluke, deposits dark brown, oval eggs with sharply defined 
operculum or cap. They gain access to the bowel by way 
of the biliary passages. The ova measure 2 0.015 to 0.017 
by 0.027 to 0.030 mm. 

Fasciola Hepatica.— Fasciola hepatica, also a liver- 
fluke, is common in many domestic animals (chiefly herbi- 
vora), though rare in man. The eggs are oval, yellowish- 
brown, with distinct operculum, and measure 0.130 to 0.145 
by 0.070 to 0.090 mm. They contain no embryo when ovi- 
posited. 

Schistosoma haematobium, the causative agent in bil- 
harziasis (venous distomatosis), inhabits the branches of 
the portal vein of man, particularly the mesenteric veins, 
and also the veins of the urinary bladder and vagina. The 
sharp-spined ova pierce the wall of the vessel, and thus it 
happens that they may be found in either urine (see p. 

1 Stiles, C. W. " Illustrated key to the trematode parasites of man. ' ' 
Bull. no. 17, U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1904. (Illustra- 
tions and full descriptions of parasites and ova are given, together with brief 
clinical notes, keys to the ova, etc.) 

2 The measurements of all trematode ova, unless otherwise indicated, are 
taken from Stiles (loc. cit.) and are the extremes reported by him from the 
literature or his own observations. 



THE FECES 



193 



118) or feces, or in both. The eggs (Fig. 26) are oval or 
spindle-shaped and measure 0.050 to 0.073 by 0.120 to 0.190 
mm. (Braun). The shell is clear, usually brown in color, 
and is provided with a sharp spine. The latter is usually 
situated laterally (subterminally) 
near one pole of the ovum when 
seen in the feces, whereas a termi- 
nal spine is commonly seen in the 
urine. The ovum contains a cili- 
ated embryo or miracidium. At 
times the latter may be seen 
swimming free in the preparation. 
The stool contains blood practi- 
cally without exception. 

Schistosoma Japonicum.— 
Schistosoma japonicum is endemic 
in Japan and in certain parts of 
China, and has been reported from 
the Philip- 
pines. It in- 
habits the 
portal and 
mesenteric 

veins chiefly ; the bladder is apparently 
unaffected. Infection of the lung (ova 
in sputum) is rare. The ova (Fig. 
27) are without spine or operculum, 
oval in shape, and measure 0.060 to 
0.090 by 0.030 to 0.050 mm. (Braun). 
fig. 27.-ovum of Scms- Each conta iiis a fully developed mira- 

TOSOMA JAPONICUM. J x 

(From a specimen pre- cidium. 

served with formalin, 

obtained through the FaSClOlopsiS BUSKll.— FaSClolopSIS 

Log^n. 8 ) 8 °X460. ' ' buskii, like Schistosoma japonicum, is 





Fig. 26. — Ovum of Schistosoma 
haematobium. (From a speci- 
men preserved with formalin.) 
X460. 



194 MICROSCOPIC EXAMINATION 

an intestinal fluke. It is widespread in Asia. The ova 
measure 0.120 to 0.130 by 0.077 to 0.080 mm. and have a 
delicate operculum. 

Paragonimus Westermanii.— Paragonimus westermanii, 
the lung-fluke, belongs to the class of parasites under con- 
sideration. Its ova may appear in the feces through swal- 
lowing of the sputa, if they pass the stomach intact. Liver 
infection has also been recorded. The eggs (Fig. 36) are 
oval, 0.068 to 0.118 by 0.048 to 0.060 mm. in size, possess 
a yellow shell, and are provided with an operculum. 

Cestodes 

Cestodes 1 or tapeworms include some of the commonest 
intestinal parasites in the United States. The larger 
worms usually disclose their presence to the infected in- 
dividual by segments, which appear in the feces. Micro- 
scopic examination of the stools oftentimes reveals ova 
where infection has not been suspected. 

Tenia Saginata.— Tenia saginata, the beef tapeworm, 
is a large parasite, measuring 4 to 10, even 36 m. in length 
when fully developed. From the mature, gravid segments 
(the segments are hermaphroditic), ova (Fig. 28, 2) are 
deposited in the feces. They are round or oval, and meas- 
ure 0.030 to 0.040 by 0.020 to 0.030 mm. (Braun). The 
shell is rather thick, radially striated, and light brown in 
color. Within it three pairs of hooklets may be visible; 
to see them it is necessary to focus carefully, as it does not 
happen often that all are in the same plane. The mature 
segments or proglottides (Fig. 28, 1) are those usually seen 
in the feces. They are 16 to 20 mm. long and 4 to 7 mm. 
broad, and are characterized by the presence of a uterus 

1 Stiles, C. W. " Illustrated key to the cestode parasites of man. ' ' Bull. 
No. 25, Hyg. Lab., U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1906. 



THE FECES 



195 



with central stem., from each side of which 25 to 30 lateral 
branches are given off; these lateral branches are them- 
selves subdivided into numerous smaller branches. The 
gross structure of the uterus may be determined by flat- 
tening the segment between two glass slides and holding 




Fig. 28. — (1) Gravid Proglottis of Tenia saginata (X4); (2) Ovum of Tenia 
faginata (X460); (3) Gravid Proglottis of Tenia solium (X4). 

it to the light. The uterus then stands out in fairly sharp 
relief. Each segment is provided with a genital pore which 
is found at one side; the pores alternate very irregularly 
from side to side. The head of the parasite is cuboidal, 
1.5 to 2.0 mm. thick. It is unarmed. 

Note. — From the ova alone it is impossible to distin- 
guish between Tenia saginata and Tenia solium (q. v.). 
The ova of Tenia saginata are quite innocuous to man, 
since the intermediary stage of the parasite, Cysticercus 
bovis, to which they give rise, develops practically only in 
beef — at all events, not in man. With Taenia solium the 
case is quite different. While the hog is the usual host of 



196 



MICROSCOPIC EXAMINATION 



the Cysticercus cellulosa?. the latter may also occur in man. 
either from the introduction of the ova or the mature. 
gravid segments into the stomach. Obviously, then, it is 
very important to handle all intestinal discharges contain- 
ing ova like those described above with extreme care, until 
the presence of Tenia solium is definitely excluded. For 
similar reasons, the patient's excreta should be thoroughly 
disinfected, preferably by burning. 

Tenia Solium.— Tenia solium, the pork tapeworm, re- 
sembles Tenia saginata in many respects. The fully grown 
parasite is 2 to 3 m. long. The ova are 
round or oval. 0.031 to 0.036 mm. in diame- 
ter (Braun). with brown shell, radially 
striated. The oncosphere is about 0.020 
mm. in diameter, and possesses six hook- 
lets. The egg is indistinguishable from 
that of Tenia saginata (Fig. 28, 2) micro- 
scopically. The mature segments (Fig. 
28, 3), which are often seen in the feces, 
are 10 to 12 mm. long and 5 to 6 mm. 
broad (Braun). They differ from the seg- 
ments of Tenia saginata in that the uterus has only 7 to 10 
lateral branches extending to either side, and they do not 
tend to rebranch. The rostellum of Tenia solium is char- 
acterized by a double crown of 22 to 32 booklets, large and 
small alternating. The head of Tenia saginata. on the 
other hand, is unarmed. 

Dibothriocephalus Latus.— Dibothriocephalus latus. the 
fish tapeworm, is the third large cestode frequently para- 
sitic in man. The mature parasite may measure 9 m. in 
length. The ova (Fig. 29) have a rather thin, clear, white 
or brownish shell, with a small operculum or cap. The 
latter stands out particularly well after treatment with 




Fig. 29. — Ovum of 

DlBOTHEIOCEPH- 
ALUS LATUS. X 

460. 



THE FECES 



197 




Fig. 30. — Gravid Proglottis of 
dlbothriocephaluslatus. x4. 



glycerin or dilute sulphuric acid (Blanchard). The eggs 
are elliptical, and present granular contents. They meas- 
ure 0.068 to 0.070 mm. by 0.044 to 0.045 mm. (Blanchard). 
The posterior segments or proglottides (Fig. 30) may be 
found in the feces, and at times are devoid of ova. Unlike 
the two preceding parasites, the 
majority of the segments are 
broader than they are long, 
though the reverse may be ob- 
served in the posterior seg- 
ments. The dark brown, rosette- 
shaped uterus, placed near the 
center of the proglottis, distin- 
guishes the parasite. The head, 
which is almond-shaped, is 2 to 
3 mm. long and is unarmed. 
Hooklets are lacking also in the ova. 

Hymenolepis Nana. 1 — Hymenolepis nana, the dwarf 
tapeworm, is a very common parasite, especially in chil- 
dren. Because of its small size, infection with this para- 
site is seldom diagnosed by the finding 
of segments in the feces. The fully de- 
veloped parasite is 10 to 45 mm. long 
and from 0.5 to 0.9 mm. thick. The head 
is round, 0.25 to 0.30 mm. in thickness, 
and presents a simple crown of 24 to 30 
hooklets. The ova are spherical or oval 
(Fig. 31). The shell is clear and trans- 
parent, at times having a light brownish 
or yellowish tint. It consists of two distinct membranes 

1 Kansom, B. H. ' ' An account of the tapeworms of the genus Hymenolepis 
parasitic in man, etc." Bull. no. 18, Hyg. Lab., U. S. Pub. Health & Mar. 
Hosp. Serv., Wash., 1904. (A full description of the parasites, with the clini- 
cal aspects of infection.) 




Fig. 31. — Ovum of 
Hymenolepis nana. 
X460. 



198 



MICROSCOPIC EXAMINATION 




Fig 



separated by an intervening space, which contains a trans- 
parent substance, more or less finely granular. At two op- 
posite points, usually corresponding to the poles of the egg y 
there is a small, mammillated projection, often not ap- 
parent. To each of these is attached a number of clear 

hyalin fibers, which pass out 
through the intermediate sub- 
stance toward the outer mem- 
brane. It frequently happens 
that the intermediate substance 
shrinks or retracts from the 
outer or inner membrane or from 
both, resulting in the appearance 
of a third membrane between the 
32.— ovum of Hymenolepis two ; in reality none exists. The 

DIMINUTA. X460. onter membrane is very thin> less 

than 0.001 mm. The inner membrane is of about the same 
thickness, and closely invests the oncosphere, which pre- 
sents three pairs of hooks, usually directed toward one 
pole (Eansom). The outer dimension of the egg varies be- 
tween 0.036 and 0.056 mm. 
long and 0.032-0.042 mm. 
broad. 

Hymenolepis Diminuta. 
— Hymenolepis diminuta, 
commonly found in rats, is 
occasionally parasitic in 
man. The parasite is 
small, being 1 to 6 cm. long 
and 2.5 to 4 mm. wide. The 
head, which is unarmed, is 
0.2 to 0.6 mm. wide. The ova (Fig. 32) resemble those of 
Hymenolepis nana. They are round or nearly so, and have 




Fig. 33. — Dipylidium caninum, showing 
an Egg Capsule and a Free Ovum. 

(After Stiles.) 



THE FECES 199 

two membranes. The outer membrane may be radially 
striated. The intervening space between the two mem- 
branes is granular. The diameter of the eggs varies be- 
tween 0.054 and 0.086 mm. (Ransom). 

Dipylidium Caninum.— Dipylidium caninum is a com- 
mon parasite in the intestines of dogs and cats ; a number 
of instances of human infection are recorded. 1 The fully 
developed parasite is 15 to 35 cm. long. The head is small 
and the rostellum club-shaped, with three to four rows of 
hooks, about 60 in number. The mature, gravid segments 
may be seen without difficulty with the unaided eye. They 
are 8 to 11 mm. long and 1.5 to 3 mm. broad; their color 
is often reddish. The genital pores are double, and are 
opposite. The ova (Fig. 33) are spherical, and have a 
diameter of 0.043 to 0.050 mm. The shell is thin. The 
uterus contains the eggs in capsules, 8 to 20 eggs being 
contained in each ; they may be encountered in the feces in 
this form (Stiles). Three pairs of hooks are to be seen 
in the oncosphere. 

Pkesekvation op Geoss Specimens of Cestodes and Othek 

Pakasites 

Stools containing parasitic ova or embryos are best 
preserved by adding commercial formalin (40 per cent.) 
to a thin watery suspension of the material, so that the 
latter contains about 2 per cent, of formalin. The major- 
ity of eggs are quite well preserved, though thin-shelled 
ova, such as those of Hymenolepis nana, show considerable 
distortion. Specimens of feces containing the rhabditiform 
embryos of Strongyloides stercoralis may be kept for sev- 
eral years; the structure may be wanting in some of the 

1 Lins, J. ' ' Sechs Falle von Taenia cucumerina beim Menschen. ' ' Wiener 
Min, Wchnschr., 1911, XXIV, 1595. 



200 PRESERVATION OF PARASITES 

embryos, but may remain characteristic in others. It is 
of interest that the formalin does not arrest the develop- 
ment of the ova of Ascaris lumbricoides. 1 The writer has 
one specimen of feces now more than four years old, pre- 
served with formalin; many of the Ascaris ova contain 
living embryos. The adult parasites may also be preserved 
in formalin (2 per cent, solution). 



Permanent Preparations of Flatworms 

For purposes of study cestode and trematode material 
may be prepared in several ways. 

(1) Method of Mink and Ebeling. 2 — The fecal material 
is mixed with physiological salt solution heated to about 
the body temperature (37° to 40° C). The worms move 
about, and the smaller, such as Hymenolepis nana, are the 
more readily seen. With forceps the parasites are trans- 
ferred to a second dish of clear salt solution, in which they 
become free of mucus and feces. They are now transferred 
to one of three solutions for fixation: (1) Alcohol, 50 to 
70 per cent., with or without glycerin; (2) Zenker's solu- 
tion (which consists of bichlorid of mercury, 5.0 gm. ; potas- 
sium bichromate, 2.5 gm. ; sodium sulphate, 1.0 gm. ; dis- 
tilled water to 1,000 c. c), or (3) a 2 per cent, formalin so- 
lution ; in any of these the material remains 14 to 16 hours. 
Zenker's fluid causes considerable shrinking and a yellow- 
ish discoloration. Formalin is best, since the natural color 

1 Morris, K. S. ' ' The viability of parasitic ova in two per cent, formalin, 
with special reference to Ascaris lumbricoides. ' ' Johns Hopkins Hosp. Bull., 
1911, XXII, 299. 

2 Mink, O. J., and Ebeling, A. H. "A method for the preparation of flat- 
worms for study. " V. S. Naval Med. Bull, 1909, III, 267. 



THE FECES 201 

of the parasite is preserved fairly well with little or no 
shrinkage. The fixative should be allowed to act not more 
than fifteen hours, when the parasites are transferred to 
the following medium: 

Syrup (glucose, 48 parts, water, 52 parts) .1,000.0 c. c. 

Methyl alcohol 200.0 c. c. 

Glycerin 100.0 c. c. 

Camphor, q. s. (to keep sterile). 

The specimens may be left in this solution indefinitely, 
though they are usually sufficiently cleared in 4 to 5 hours. 
The material is now placed on a slide in glycerin jelly. 
After the latter has hardened (24 hours or more), the cover 
glass is sealed with cement. 

(2) Boggs' Method. 1 — The worm is washed free of 
feces, and is placed in water or salt solution, in which it 
is allowed to die, so that it may be fixed while relaxed. It 
is then placed in a solution of 20 per cent, glycerin in 80 
per cent, alcohol, which both fixes and clears the specimen. 
It is allowed to remain in this fluid in a partially covered 
dish until the alcohol is entirely evaporated. The specimen 
is then clear. It is transferred to a glass slide, and the 
excess of glycerin is removed by blotting paper. Glycerin 
jelly is then placed on the specimen, which is covered with 
a cover glass. In 24 to 48 hours, after the jelly has solidi- 
fied, the preparation is sealed with microscopical cement. 
To prevent curling of the specimen, it may be spread on 
a piece of heavy filter paper before immersing it in the 
glycerin-alcohol solution ; it may be necessary to put a light 
weight on the cover slip until the jelly hardens. 

1 Boggs, T. E. Personal communication, and in Emerson, C. P. " Clinical 
Diagnosis." 3rd Ed. Philadelphia and London, 1911, p. 444. 



THE FECES 203 

Glycerin jelly is prepared as follows : 

Gelatin (gold mark) 14.0 gm. 

Distilled water (boiling) 120.0 c. c. 

Dissolve in the hot water and add — 

Glycerin 120.0 c. c. 

Cool to 50° C. and add the whites of two eggs. Heat gently 
without stirring. Strain the mixture through a fine-meshed 
wire sieve, and filter through cotton while still warm. Add 
water to make the volume 240 c. c. and 1 c. c. of pure car- 
bolic acid as a preservative. The jelly solidifies on cool- 
ing. For use, melt it by immersing the flask containing it 
in hot water. 

(3) Creosote Method.— The material is placed in 70 
per cent, alcohol, and then in 95 per cent, alcohol, for 15 
to 30 minutes. It is then transferred to Beechwood creo- 
sote, in which it remains until the tissue is cleared. The 
time required varies with the size of the specimen and with 
the degree to which water was withdrawn by the alcohol. 
Finally, the specimen is mounted in balsam. 

Accidental contaminations through food or drink may 
account for some of the ova found in the feces. As an ex- 
ample, the ova of the Tyroglyphus siro (Fig. 34) may be 
cited. This is the common cheese-mite, which may also 
be found in flour and other articles of diet. The mites may 
be found in the stool in addition to the ova. Measurements 
of the ova may serve to differentiate them from those of 
intestinal parasites. In cases of doubt, it is advisable for 
physicians to submit the material to a zoologist for deter- 
mination. Such examinations are made at the Hygienic 
Laboratory, U. S. Public Health Service, Washington, D. C. 



CHAPTER IV 
THE SPUTUM 

In the strict sense of the word, sputum refers to the 
expectorated material which arises in the respiratory pas- 
sages between the lung alveoli and the larynx. 

To obtain sputum for examination, the patient should 
be told to discard the nasal and pharyngeal discharges. He 
should be instructed as to the proper receptacle. In case 
the physician does not supply a sputum box or cup, the 
patient may conveniently use a wide -mouthed bottle, which 
has been thoroughly cleansed and sterilized by boiling. In 
cases where a sputum examination is urgently indicated 
but no sputum is expectorated, expectorants, such as am- 
monium chlorid. may be given. Hausmaun * advises that 
the fasting stomach be washed out in the early morning 
with a view to obtaining bronchial mucus ; he reports valu- 
able findings with this method. TVith children it is not 
infrequently necessary to wash out the stomach, examine 
the feces, or place the finger, covered with gauze, in the 
child's throat after a coughing spell and mop out the 
sputum. 

The importance of repeated examinations of the sputa 
cannot be overemphasized. Particularly when looking for 
the tubercle bacillus, if the physical signs or history are 
even suggestive, examinations should be continued. A 
single negative result means nothing. 

1 Hausmann. T. " Die Frith diagnose der Lungentmberkulose durch die 
Mageninhaltsuiitersiichuiig. ' • Deutseh. Arch. f. Uin. Med.. 1908, XC1V, 595. 

201 



THE SPUTUM 205 

Amount.— The quantity of the sputum expectorated in 
twenty-four hours varies greatly in disease. An approxi- 
mate estimate of the amount is usually sufficient. 

Reaction.— The reaction of fresh sputum is usually al- 
kaline. An old specimen or sputum which has stagnated 
in the body may be acid in reaction. 

Character.— Sputum is designated mucoid, mucopuru- 
lent, purulent, serous, or bloody, as the case may be. The 
terms are self-explanatory. Various combinations are met 
with. Bloody sputa may assume any of the shades seen in 
a bruise. In the presence of jaundice, it must be remem- 
bered that green sputa do not necessarily indicate a pre- 
vious pulmonary hemorrhage; the color is in most cases 
simply a manifestation of the icterus. Bacterial growth 
may at times account for a green color. 

Odor.— The odor of sputa is important chiefly in con- 
nection with putrid affections of the bronchi or lungs. 

Consistence.— The consistence of the sputum is gener- 
ally dependent on the quantity expectorated. With large 
quantities, the consistence is usually thin, though the sputa 
of croupous pneumonia form a notable exception. Simi- 
larly, when the sputum is small in amount, it is usually 
more or less tenacious. 

Air Bubbles.— Most sputa contain air bubbles. The size 
of the bubbles is said to indicate roughly the caliber of the 
bronchi from which the expectorated material is derived. 
More air is contained in sputum from the smaller bronchi 
than from the large. 

Dittrich's Plugs.— Dittrich's plugs are sausage-shaped 
casts of the bronchi, varying in size up to that of a white 
bean. Microscopically, they may be seen to contain fat 
droplets, fatty acid crystals, cell detritus, and bacteria. 
A few pus cells and occasionally red blood corpuscles or 

15 



206 MICKOSCOPIC EXAMINATION 

hematoidin in granules or needles may be observed, less 
commonly flagellates. 

Bronchial Casts.— Bronchial casts are observed in some 
diseases with considerable frequency. Their size is deter- 
mined by that of the bronchi giving rise to them. Casts 
are usually branched, and consist mainly of fibrin, in which 
leukocytes, red blood cells, epithelial cells, etc., may be em- 
bedded. If the casts are small, their isolation is facilitated 
by placing the sputum on a white plate, half of which has 
been painted black. By teasing the specimen with needles, 
the casts may be found, provided they are macroscopic in 
size. The addition of water often renders the teasing 
easier. 

Curschmann's Spirals.— Curschmann's spirals occur in 
various sizes. Only the larger specimens are visible to the 
unaided eye. The largest spirals measure about 1 mm. in 
thickness, and are ten to twenty-five times as long. They 
can only be distinguished as spirals by microscopic exam- 
ination. 

Layer Formation.— When abundant, as in the case of 
sputa from bronchiectatic cavities, for example, distinct 
layer formation may be observed. Solid particles collect 
at the bottom, then a fluid portion, with frothy mucus on 
the surface. Frequently strands of mucus dip down from 
the upper layer. 

MICROSCOPIC EXAMINATION 

Examination of the Fresh Sputum.— Examination of the 
fresh sputum is greatly neglected. It is as important in 
the routine study of sputa as the various staining methods, 
and may furnish information which can be gained in no 
other way. 



THE SPUTUM 207 

For the examination of the fresh specimen two glass 
plates are required, one about the size of the stage of the 
microscope or a little smaller, the other of the same length 
or even a bit longer, but about one inch narrower. Old 
photographic plates, when cleansed, answer the purpose 
very well ; they may be cut into any size desired. In place 
of the second plate, a glass slide (3x1 in.) may be used. 
For handling the sputum steel hat-pins are useful. They 
are inexpensive, and are easily sterilized by heating in the 
flame of the Bunsen burner. 

Some of the sputum to be examined is transferred to 
the larger plate by means of the hat-pins. It should be 
so placed on the plate that, when spread out, all parts of 
the specimen will be accessible for microscopic examina- 
tion. After sterilizing the hat-pins * the second plate or 
the glass slide is placed over the sputum, which is spread 
out in a thin layer. Examination is made with the low 
power objective, for the thickness of the upper plate is 
usually greater than the working distance of the higher 
power dry objective. Furthermore, higher magnification 
is often unnecessary. The magnification may be increased, 
however, by selecting a strong ocular or by drawing out 
the tube of the microscope. 

After spreading the sputum between the two plates, it 
is examined macroscopically on a dark background, and 
any small, opaque masses are noted and singled out for 
microscopic inspection. Examination then determines 
whether the particle should be transferred to a slide and 
studied under a cover glass with higher magnification, or 
used for staining, or for both purposes. Curschmann's 
spirals, necrotic tissue fragments, etc., are looked for in 

1 All sputa should be considered as infectious material, and handled ac- 
cordingly. 



208 MICROSCOPIC EXAMINATION 

this way. The macroscopic examination should always be 
made, for a great deal of time may be wasted, to say noth- 
ing of overlooking important findings, if the microscopic 
examination is performed aimlessly. There are, of course, 
specimens in which macroscopic examination reveals noth- 
ing, where microscopic study of the preparation is fruitful. 
But in the majority of instances the naked eye inspection 
of the thinly spread specimen is an important adjuvant to 
microscopic examination. 

Yellow Elastic Tissue.— Elastic tissue fibers are most 
easily found by means of the glass plate method. If pres- 
ent, they are often found in yellowish, opaque masses of 
necrotic tissue about the size of a pinhead or thereabouts. 
The fibers may present roughly the outline of one or more 
alveoli (alveolar elastic tissue), single fibers or several 
long fibers forming a network may be seen (bronchial), or 
there may be sheets of elastic tissue (arterial). Often only 
isolated fibers are met with. The yellow elastic fibers are 
readily seen, and are characterized by (1) their uniform 
diameter, (2) sharp outline, (3) great refractivity, (4) curl- 
ing ends, (5) a tendency to branch, and (6) by the fact that 
pressure on the slide produces no varicosities or thicken- 
ings in the fibers. 

Fatty acid crystals, which may be mistaken for elastic 
tissue, usually present varicosities after pressure, and are, 
furthermore, unlike elastic tissue in that they are soluble 
in ether or KOH. Again, warming the preparation trans- 
forms fatty acid needles into droplets. In contrast to the 
wavy elastic tissue fibers, fatty acid crystals usually pre- 
sent a single curve. 

Curschmann's Spirals.— Curschmann's spirals, often 
visible macroscopically but never recognizable as such, 
stand out with distinctness on microscopic examination. 



THE SPUTUM 209 

They occur in two forms ; in the one there is seen a twisted 
spiral consisting of delicate, thread-like filaments of mucus, 
in the turns of which eosinophilic leukocytes, pus cells, epi- 
thelial cells, Charcot-Leyden crystals, etc., are caught; in 
the other form there is a highly refractive central filament, 
about which the mantle of mucus containing eosinophiles, 
etc., is twisted. The spirals are subject to much variation 
in size. Isolated central filaments may be found. The 
thickness of the filaments differs in different spirals, but 
in a given specimen it is quite uniform. The finest fila- 
ments are extremely minute, while the largest may be twice 
as thick as a red blood corpuscle. 

Fairly satisfactory permanent preparations of spirals 
may be had by mounting them in glycerin jelly and sealing 
the specimen with cement after the jelly has hardened. 

Dust cells, "heart failure" cells, Charcot-Leyden crys- 
tals, and other objects may be seen on examining the spu- 
tum on the glass plate. The Charcot-Leyden crystals may 
be so small as to escape detection. It may, therefore, be 
advantageous to use a higher magnification in their study. 
For this a selected particle of sputum is transferred to a 
glass slide and pressed out under a cover glass. 

Alveolar epithelial cells, derived from the lung alveoli, 
are constantly present in the sputum. Their shape varies 
greatly, since they are possessed of ameboid motion — a 
fact which is readily demonstrable by examining a perfect- 
ly fresh specimen on a warm stage. They are relatively 
large cells, but are not of uniform size. The nucleus is 
large and oval. The protoplasm of the alveolar cells is 
rather coarsely granular, but soon undergoes degeneration, 
as a result of which fat droplets and myelin granules make 
their appearance in it. 

The fat droplets in alveolar cells are, like similar drop- 



210 MICROSCOPIC EXAMINATION 

lets elsewhere, of all sizes, usually round, refractive, and 
slightly greenish, especially the larger drops. Their na- 
ture is determined by adding a drop of alcoholic solution 
of Sudan III or Scharlach E to the specimen, by means of 
which fat is stained orange or orange-red. 

Myelin degeneration of the alveolar cells gives rise to 
the macroscopic masses in sputa resembling boiled sago. 
The myelin droplets may be large or small, and, un- 
like fat droplets, they are often quite irregular in con- 
tour. At times the center of a mass of myelin appears to 
be thinned. Myelin granules are refractive and have a 
greenish tint, which is more pronounced than that seen in 
fat droplets. Myelin is frequently found free in the spu- 
tum, probably the result of disintegration or mechanical 
rupture of the degenerated epithelial cells. It does not 
take the fat stains. 

Dust cells are found in the sputum of all who inhale 
coal dust. They are alveolar epithelial cells, which have 
phagocyted the minute particles of coal dust, which con- 
stantly are inspired in a smoky atmosphere. The dust 
appears as dark, brownish-black spots in the cell, which 
may be so heavily laden that nucleus and protoplasm are 
entirely obscured. Dust cells are easily distinguished in 
examining sputum by the plate method. When they are 
numerous, the sputum is stained more or less diffusely 
black. 

" Heart-failure cells" are alveolar epithelial cells which 
have taken up blood pigment. The name is a misnomer, 
for they appear in the sputum after a pulmonary or bron- 
chial hemorrhage from any cause whatever. The pigment, 
which is hematoidin, appears as light, golden-yellow gran- 
ules, which can scarcely be confused with coal dust. 

Red blood corpuscles are often seen in a state of preser- 



THE SPUTUM 211 

vation. If the hemorrhage is an old one, however, the cells 
disintegrate, and only hematoidin in amorphous masses — 
usually in heart-failure cells — or in needles will be discov- 
ered. 

Pus cells, polynuclear neutrophilic leukocytes, are al- 
ways present in the sputa microscopically. The polymor- 
phous nucleus in a cell about 12 micra in diameter with 
finely granular cytoplasm is characteristic. Occasionally 
fat droplets are contained in the cells, or they may take 
up foreign particles in the air passages. In a fresh speci- 
men active ameboid movements may be observed in these 
cells; pseudopodia are protruded, and the granules of the 
protoplasm are actively motile. 

Eosinophilic leukocytes, of the same size as pus cells 
and having a polymorphous nucleus, are distinguished by 
their protoplasmic granules. The latter are coarser than 
the neutrophilic granules, and are highly refractive, glis- 
tening bodies. Ameboid motion may also be observed in 
these cells. Free granules are usually present in the speci- 
men. 

Lymphocytes, or cells which are identical morphologi- 
cally, are present at times in large numbers. The round or 
oval nucleus with narrow rim of protoplasm and the small 
size of the cells — 7 to 12 micra — together with the non- 
granular cytoplasm are distinctive. 

Charcot-Leyden crystals are usually found in the spu- 
tum with large numbers of eosinophile cells. They are 
formed wherever eosinophile cells disintegrate. In form 
the crystals are long lozenges. Their edges are clean cut, 
the points sharp, and the crystals have a yellowish or 
greenish tint. They are quite fragile, and the larger crys- 
tals may be broken in preparing the specimen. They oc- 
cur singly or in clusters, and vary greatly in size; the 



212 MICROSCOPIC EXAMINATION 

smallest are visible only with the oil-immersion objective, 
while large crystals are seen without difficulty with low 
magnification. On cross-section the crystals are hexagonal. 
Like the eosinophilic granules, they may be stained with 
eosin. They are soluble in mineral acids, alkalies, and 
boiling water. The crystals may be apparently lacking in 
sputa which contain enormous numbers of eosinophiles. 
This is particularly apt to be the case when a sputum is 
first flooded with these cells. If examinations are made 
from day to day the crystals are found sooner or later. 

Crystals of fatty acid, cholesterin, hematoidin, triple 
phosphate, etc., may be encountered in sputa, especially 
after stagnation. (For morphology and microchemical re- 
actions see the chapter on the urine or feces.) 

Microorganisms in Sputa 

Only the more important microorganisms of the sputum 
are referred to in the following pages, and in no case arc 
cultural methods described. For this and details of mor- 
phology the reader is referred to works on bacteriology. 

Bacillus Tuberculosis 

Bacillus tuberculosis may be found in any kind of spu- 
tum, for the gross appearance of the sputum in pulmonary 
tuberculosis is in no way distinctive; it may, in fact, be 
anything. If the plate examination shows necrotic tissue 
(elastic tissue), it should be selected for staining, as the 
bacilli are generally more numerous in such material. Oth- 
erwise purulent particles are most suitable for examina- 
tion. 

The preparation for examination is made by smearing 
the selected particle on a glass slide with a hatpin or other 



THE SPUTUM 213 

suitable object. Or it may be pressed between two slides, 
which are then drawn apart, so that the material is smeared 
in a thin layer on each of them. In the second way prep- 
arations of more uniform thickness are obtained, but there 
is usually some of the material at the edge of the slide, with 
which the fingers or other objects may become contami- 
nated. (This is referred to not because it is a valid objec- 
tion to the method, but because the writer has so frequently 
observed carelessness in this particular point. Still, one 
who neglects such an obvious source of infection is sure to 
make other more serious breaks in technique, and has no 
business examining infectious material, both for his own 
safety and, more particularly, for the safety of others.) 
If the sputum dries slowly, it may be hastened by warming 
the slide gently. The smear is then fixed in the usual way 
by passing it through the flame of the Bunsen burner sev- 
eral times. 

Ziehl-Neelsen Method.— The Ziehl-Neelsen method of 
staining is the one generally employed for the tubercle 
bacillus. The reagents required are: 

(1) Carbol-fuchsin. 

(a) Fuchsin 1.0 gm. 

Absolute alcohol 10.0 c. c. 

Dissolve and add — 

(b) 5 per cent, carbolic acid 100.0 c. c. 

(2) Acid alcohol. 

Hydrochloric acid, cone 3.0 c. c. 

70 per cent, alcohol to 100.0 c. c. 

(3) Loffler's methylene blue. 
Methylene blue, saturated alcoholic 

sol 30.0 c. c. 

0.01 per cent, potassium hydrate. . .100.0 c. c. 



214 MICROSCOPIC EXAMINATION 

Method. — (1) Cover the specimen with carbol-fuchsin 
and warm it till the stain steams. Maintain this tempera- 
ture for five minutes. 1 Or the specimen may be immersed 
in the cold stain for twenty-four hours. It is important 
to overstain the preparation with carbol-fuchsin, for at 
best many of the bacilli are decolorized. TVith light stain- 
ing, when only a few bacilli are present, they may be missed 
entirely (L. Brown). 

(2) Eemove the excess of stain by washing in running 
water. 

(3) Decolorize in acid alcohol, until only the thickest 
parts of the smear have a faint pinkish tint. 

(4) Again wash in water. (Eeturn to the acid alcohol 
if the specimen becomes pink after washing.) 

(5) Stain with Lomer's methylene blue 5 to 20 seconds. 

(6) Wash in water, dry the preparation in the air or 
between sheets of blotting paper. Examine in immersion 
oil. 

The tubercle bacilli are stained red ; all else is blue. 

Antiformin Method for the Detection of Tubercle Ba- 
cilli.— In 1908 TThlenhuth and Xylander 2 made the impor- 
tant discovery that antiformin possesses the peculiar prop- 
erty of dissolving all bacteria except those which are acid- 
fast, to which class the tubercle bacillus belongs. Applied 
to the sputum, they found that, in addition to the major- 
ity of bacteria, the great mass of the sputum is also lique- 
fied. By the use of antiformin it is, therefore, possible to 
examine a large quantity of sputum and thus to concen- 
trate the tubercle bacilli present in it. The method is val- 

1 The copper bar used in blood "work is convenient for heating the specimen. 
The stain must be replenished from time to time as it evaporates, to prevent 
burning the specimen. 

- Uhlenhuth and Xylander. "Antiformin, ein bakterienauflosendes Desin- 
f ektionsmittel. ; ; Berlin. TcJin, Wchnschr., 1908, XLV, 1346. 



THE SPUTUM 215 

uaMe in those cases where the ordinary technique fails to 
demonstrate bacilli. A number of methods have been de- 
scribed for the use of antiformin, of which the following 
have been found serviceable: 

(1) Loffler 's Method. 1 — The quickest method for the 
use of antiformin with sputum, and one which is well 
adapted to clinical work, has been described by Loffler. 
With this procedure the examination may be completed in 
a comparatively short time. A quantity of sputum (5 to 
20 c. c.) is measured, placed in a beaker or flask of Jena 
glass with an equal volume of 50 per cent, antiformin, and 
boiled. Solution of the sputum occurs almost at once, the 
fluid foaming and turning light brown. To 10 c. c. of the 
cooled solution, which is sterile, add 1.5 c. c. of a mixture 
composed of 1 volume of chloroform and 9 volumes of al- 
cohol. After shaking thoroughly, the specimen is centrifu- 
galized for about fifteen minutes (the time varies with the 
speed of the centrifuge). The chloroform is thrown to the 
bottom of the tube, and on its surface the sediment col- 
lects. The supernatant fluid is poured off and the sediment 
transferred with a clean pipette to a glass slide. The ex- 
cess of fluid is removed with filter paper, and a small drop 
of egg albumin (which may be preserved by the addition 
of 0.5 per cent, carbolic acid) is mixed with the sediment, 
which is spread on the slide, fixed, and stained in the usual 
manner. The original sputum may be substituted for egg 
albumin as the fixative; it is, indeed, preferable, since a 
more complete examination of the sputum is possible. 

The tubercle bacilli are said to be killed with this method 
(Loffler). 

1 Loffler, F. ' ' Ein neues Anreicherungsverf ahren zum f arberischen Nach- 
weise sparlicher Tuberkelbazillen. ' ' Deutsche med. Wchnschr., 1910, XXXVI, 
1987. Also Williamson, C. S. "The value of the Loeffler method of sputum 
examination." Jour. A. M. A., 1912, LVIII, 1005. 



216 MICBOSCOPIC EXAMINATION 

As a counterstain Loffler uses malachite green (0.1 per 
cent, aqueous solution). 

(2) Pateeson's 1 Method. — Paterson adds to 10 c. c. of 
sputum 2.5 c.c. of antiforniin, 2 giving a 20 per cent, strength 
of the latter. If the sputum is very thick or tenacious, or 
insufficient in quantity, a smaller amount is diluted to 10 
c. c. with distilled water. 3 Solution of the sputum occurs 
rapidly. The mixture is poured into centrifuge tubes, 
which have been kept in potassium bichromate and sul- 
phuric acid, 4 and rinsed with distilled water just before 
using. The tubes are stoppered with unused corks, shaken 
vigorously, and set aside for 24 hours at room temperature 
or for 4 to 6 hours at 37° C. The tubes are again shaken 
and then centrifugalized. The supernatant fluid is poured 
off, the tubes refilled with sterile physiological salt solu- 
tion, again corked, shaken, and centrifugalized. The wash- 
ing is done a second time to rid the sediment of all alkali ; 
otherwise it does not adhere well to the glass. The sedi- 
ment is then transferred to a slide, smeared, fixed, and 
stained by the Ziehl-Xeelsen method. 

Paterson, R. C. "A report on the use of 'antiformin' for the detection 
of tubercle bacilli in the sputum, etc." Jour. Med. Research, 1910, XXII, 315. 

2 The composition of antiformin, according to Paterson, is equal parts of 
15 per cent, solution of sodium hydrate and of liquor sodae chlorinatse (B. P.). 
The latter is prepared as follows: 

Sodium carbonate 600.0 gm. 

Chlorinated lime 400.0 gm. 

Distilled water 4,000.0 c. c. 

Dissolve the sodium carbonate in 1.000 c.c. of distilled water. Triturate 
thoroughly the chlorinated lime in the remainder of the water. Filter. Mix 
the two and filter again. There is formed an alkaline, almost colorless liquid 
with a strong odor of chlorin. It keeps well. 

'Before this is done, the existence of acid-fast bacilli in the distilled 
water should be excluded — a troublesome source of error at times, as shown by 
W. Brem (Jour. A. M. A., 1909, LIII, 909). 

4 Rub up some potassium bichromate with sulphuric acid for two minutes, 
allowing the acid to take up as much of the bichromate as it will. Pour off the 
acid and repeat the process. 



THE SPUTUM 217 

The washing with salt solution may be dispensed with. 
The sediment, which consists of debris, swollen and dis- 
torted cells, etc., is fixed to the slide with difficulty because 
of the alkali, but this may be overcome by first smearing 
the slide with some of the original sputum or with egg 
white (preserved by the addition of 0.5 per cent, pure car- 
bolic acid). The original sputum is to be preferred, since 
the other elements present in it may thus be studied. 

The tubercle bacilli are not killed and the sediment 
should, therefore, be handled with the usual precautions. 

The method is valuable for obtaining material for ani- 
mal inoculation or for cultures. 

(3) Boakdman's Method. 1 — The following procedure 
has been found satisfactory by Boardman: Fifteen to 20 
c. c. of sputum are placed in a conical specimen glass, and 
antiformin is added sufficient to make a 20 per cent, 
strength of the latter. After the solution has become homo- 
geneous and watery in consistence, an equal volume of 95 
per cent, alcohol is added. By this means sedimentation is 
facilitated, since the specific gravity of the mixture is less 
than 1.000. After stirring, allow it to stand till sedimenta- 
tion is complete. The clear supernatant fluid is poured off 
(into a disinfecting solution), and smears are made of the 
sediment on a glass slide, using some of the original sputum 
as a fixative. The specimen is then fixed with heat and 
stained "in the usual way. 

Diplococcus Pneumonia 

The pneunio coccus, found not infrequently in the upper 
respiratory passages of healthy individuals, is often con- 

1 Boardman, W. W. ' ' The use of antiformin in the examination of the 
sputum for the tubercle bacillus.' 7 Johns Hopkins Hosp. Bull., 1911, XXII, 
269. 



218 MICROSCOPIC EXAMINATION 

spicuous in the sputum of acute lobar pneumonia and other 
conditions. It is a Gram-positive organism, whose capsule 
may be seen after staining with Gram's method. 

Gram's Method of Staining. 

Eeagents : 1. Anilin water gentian violet. 

Ten c. c. of anilin oil are shaken with 100 c. c. of dis- 
tilled water till a milky emulsion is secured. After stand- 
ing five minutes, it is filtered through a wet filter. To the 
filtrate, which should contain no large oil drops, add 11 
c. c. of saturated alcoholic solution of gentian violet and 
10 c. c. of absolute alcohol. The solution keeps not more 
than 8 to 10 days (Schmorl). 

2. Gram's iodin solution: 

Iodin 1.0 gm. 

Potassium iodid 2.0 gm. 

Distilled water 300.0 c. c. 

Method. — (1) Stain the heat-fixed smear in anilin water 
gentian violet 1 to 3 minutes. 

(2) Wash quickly in water. 

(3) Cover the specimen with Gram's iodin solution 
about 1% minutes. 

(4) Decolorize in absolute alcohol until the preparation 
has a grayish or yellowish color — usually about 5 minutes. 
(The specimen may now be dried and examined. If a 
counterstain is desired, the further steps are carried out.) 

(5) Wash in water. 

(6) Stain with 0.2 per cent, aqueous solution of Bis- 
marck brown 1 minute. 

(7) Wash in water, dry in the air or blot, and examine 
in immersion oil. 

The pneumococci and all other Gram-positive organisms 



THE SPUTUM 219 

are stained blue, the remaining bacteria and cell nuclei 
are brown. 

The pneumococcus or Diplococcus pneumoniae grows in 
pairs. The long axes of the organism are placed end to 
end. 

For demonstration of the capsules, Welch's method 
may be employed. 

Welch's Capsule Stain.— (1) Flood the fixed smear with 
glacial acetic acid, and immediately pour it off. 

(2) Wash off the acid with anilin water gentian violet. 

(3) Wash in 2 per cent, sodium chlorid solution, and 
examine the wet specimen. 

Bacillus Influenza 

Bacillus influenza is a very minute bacillus, which often 
exhibits polar staining. It may be found free in the spu- 
tum or within pus or epithelial cells. It decolorizes by 
Gram's method of staining and, therefore, takes the coun- 
terstain. It may be stained satisfactorily with carbol-fuch- 
sin. The stain is diluted 1:10 with distilled water, and 
allowed to act for ten minutes or longer. Cultural methods 
are required for the complete identification of Bacillus in- 
fluenzae. It grows on media containing blood. 

Bacillus Diphtheria 

Bacillus diphtheria, though not usually found in the 
sputum, may be considered here. In examining for it the 
membrane is brushed with a sterile swab, with which, after 
inoculating tubes of Loffler's serum, smears may be made 
on glass slides. The smears may show the organism, but 
in any case it is better to examine a culture on blood serum 
which has been incubated at body temperature for 9 to 18 



220 MICROSCOPIC EXAMINATION 

hours. From a fresh culture, of the age given above, the 
organisms exhibit polar staining. Usually two deeply 
staining bodies are seen in a bacillus at either pole, though 
there may be but one or as many as three. Chains are for 
the most part lacking; frequently the bacilli are pla 
with their long axes parallel. The polar staining is beau- 
tifully demonstrated with Neisser's method. 
Neisser's Staining Method. 

Eeagents : 

(1) Methylene blue 1.0 gin. 

Alcohol, 90 per cent 20.0 c. c. 

Dissolve and then add — 

Distilled water 950.0 c. c. 

Acetic acid, glacial 30.0 c. c. 

(2) Vesuvin (Bismarck brown) 2.0 gin. 

Boiling distilled water 1.000.0 c. c. 

Dissolve. Filter after the solution cools. 

Method. — (1) Stain in methylene blue solution 1 to 3 
seconds. 

(2) Wash quickly in water. 

(3) Stain in vesuvin 3 r 5 seconds. 

(4) Wash quickly in water, blot dry. and examine in 
oil. 

The diphtheria bacilli are slender rods, often having a 
slight bend. They are stained light brown, with one to 
three granules, which take a dark blue color. Parallel 
pairs of bacilli are characteristic. Chain formation is 
lacking. Other bacteria, which are present in the smear, 
are stained light brown, so that the blue polar bodies of 
Bacillus diphtheria? are striking and characteristic. 



THE SPUTUM 221 

Beall's Method. 1 — The polar bodies may be demon- 
strated well by Beall's method. 

(1) Over stain the specimen with anilin water gentian 
vdolet % to y 2 minute. 

(2) Wash in water. 

(3) Decolorize with 10 per cent, glacial acetic acid till 
little color remains. This is controlled under the micro- 
scope. 

(4) Wash in water, blot dry, and examine in immersion 
oil. 

All Gram-negative organisms are decolorized, and most 
of those which are Gram-positive. The polar staining is 
intense, and diphtheria bacilli stand out prominently. 
Practically all other organisms are decolorized more or 
less completely. 

Actinomyces Bovis 

Actinomyces bovis (Fig. 35), the ray fungus, which 
is the causative agent in "lumpy jaw" of cattle, is 
occasionally parasitic in man. The lungs are often the 
site of infection. 2 The sputum is characteristic. Claypole 
studied the series of cases reported by Bridge, and de- 
scribes the sputum as follows: "In the majority of cases 
the sputum is characteristic and of two types: (1) glairy, 
mucilaginous, often quite watery; (2) purulent, more or 
less bloody, more or less — sometimes intensely — fetid. 
Both types may be found sparingly or in abundance. 
. . . The small granules (of the fungus), usually the 
size of a very small pinhead, can be picked out with a 

1 An unpublished method of Dr. H. K. Beall of Fort Worth, Texas, 
through whose kindness it is given here. 

2 For a review of the subject, see Bridge, N. ' ' Streptothricosis (actino- 
mycosis) of the lungs. ' ' Jour. A. M. A., 1911, LVII, 1501. 

10 



000 



MICROSCOPIC EXAMINATION 



c- 



needle and put on a slide for examination. They are quite 

tough, and can be washed free of debris by putting them 

in a dish of water and squirting them vigorously up and 

down a pipette. ... " 

"Under low magnification the yellow color is marked: 

to the naked eye the fungus is grayish-white. The edge 

is always darker, even shading 
into a brown; toward the center 
it grows lighter. From this light, 
almost homogeneous center, th? 
characteristic radiations arise. 
Higher magnification shows the 
center to be a mass of pale, radi- 
ating threads, the mycelia. and at 
the edges a mass of threads and 
cocci. Both mycelia and cocci 
may be stained with methylene 

blue, the former frequently being banded light and dark in 

segments,, sometimes granular throughout." 




Fig. 35. — Actts-omtces homtnts. 
Showts-g Club-shaped Ex- 
tremities TO THE R_4.YS. 
(Fresh preparation.) (After 
Wood.) 



Streptoth rix Eppengeri 



Streptothrix eppengeri is an organism related to the 
preceding. In the case described by \Varthin and Olney 1 
a filamentous, branching organism was found. Usually the 
mycelia were tangled and interwoven. No conical or club- 
shaped terminations were found, as in Actinomyces bovis. 
The threads, stained with carbolfuchsin. were not decol- 
orized after treatment with 25 per cent, nitric or sulphuric 
acids, though the stain was largely removed by washing in 
95 per cent, alcohol. 

1 Warthin. A. S.. and Olney. H. S. " Pulmonary streptothricosis. ' ' Amer. 
Jour. Med. Sci., 1904, CXXTXEI. 637. 



THE SPUTUM 



223 



Blast omycetes 

Blastomycetes (Fig. 36) have been found in the spu- 
tum in a number of cases. "In unstained preparations of 
pus and tissue the organisms appear as round or oval 




Fig. 36. — Blastomycetes in Sputum. X 1500. Photomicrograph, (After E. E. 
Irons and E. A. Graham.) 

bodies with a double contoured, highly refractive capsule. 
Within the capsule, in many instances, granules or spore- 
like bodies can be distinguished. The addition of a 1 to 
10 per cent, solution of potassium hydrate to the specimen 



224 MICROSCOPIC EXAMINATION 

under examination facilitates the recognition of these 
bodies. In stained sections the double-contoured, homo- 
geneous capsule is usually separated from a finely or 
coarsely granular protoplasm by a clear space of varying 
width. Vacuoles of different sizes are found in some or- 
ganisms. In both pus and tissue organisms in pairs or in 
various stages of budding are commonly seen. The para- 
site, as a rule, varies in size from 7 to 20 micra, though 
slightly smaller and much larger forms occur in some 
cases." 2 

Animal, Paeasites in the Sputum 

In this country animal parasites are comparatively rare 
in the respiratory passages, though probably more common 
than is generally supposed. 

Entameba histolytica (Pig. 12) may be encountered 
in the sputum as the result of rupture of 
an amebic liver abscess into the bronchial 
tree. The organism is identical with that 
found in the feces (q. v.). Perfectly fresh 
sputum should be examined — when possi- 
ble, with a warm stage. 
fig. 37. — ovum of Entameba tetragena (Fig. 12) occurs 

Paragoximus wes- m the sputum under the same conditions 

TERMAXII FROM THE x 

Sfdtdm. X400. as Entameba histolytica. 

(After Emerson.) . . , n 

Tnchomonads (Fig. 13) and cerco- 
monads are occasionally found in the sputa, usually in ma- 
terial which has stagnated in the lung. 

Paragonimus westermanii, the lung-fluke, the cause of 
"parasitic hemoptysis," is rare in this country, common 

1 Montgomery. F. H. ( and Orinsby, 0. S. "Systemic blastomycosis."' 
Arcli. Int. Med., 190S, II, 1. See also Hektoen, L. " Systemic blastomycosis 
and coccidioidal granuloma. '" Jour. A. If. A.. 1907, XLIX, 1071. (Litera- 
ture.) 



THE SPUTUM 225 

in Japan and parts of China. Diagnosis is made by find- 
ing the ova (Fig. 37) in the fresh sputum (see p. 194). 

Echinococcus cyst, though common in certain parts of 
the world, is excessively rare in this country. Diagnosis 




Fig. 38. — Sediment from Echinococcus Cyst. Above and to the left are two 
degenerated seolices (X about 60); to the right is a crown of hooklets (X400); 
below are hooklets of unusual shapes and a small mass of cholesterin crystals 
(X400). (After Emerson.) 

may be made after rupture of the cyst into the bronchi 
by (1) the finding of daughter cysts, (2) seolices, (3) hook- 
lets, or (4) parts of the membrane in the sputum (Fig. 38). 
The material may arise from a hepatic cyst which has rup- 
tured through the diaphragm into the air passages. 



CHAPTEE V 

THE BLOOD 

Obtaining Blood for Examination.— Blood for counts, 
etc., is obtained most conveniently from the lobe of the 
ear or the ball of the finger. The ear is, on the whole, more 
satisfactory than the finger. It is easily accessible, the flow 
of blood is as good, and it is much less sensitive to pain 
than the finger. There is also less likelihood of infection 
of the small wound through contact with dirty objects. For 
counts or hemoglobin determinations, blood should not be 
drawn from a part of the body which is cyanotic, because 
concentration of the blood may occur, producing results 
which are misleading (too high). 

Blood Stickers.— A number of satisfactory blood stick- 
ers are on the market, and require no special description. 
In default of these a Hagedorn needle may be used. Bass 1 
has recently described a simple arrangement consisting of 
a straight surgical needle mounted in a cork. TThen not 
in use, the needle is carried in a small vial filled with al- 
cohol. A sharp steel pen. one of whose prongs has been 
broken off. may be employed as a sticker. The sticker 
should always be perfectly clean, in addition to being ster- 
ile. Dried blood on the point, even a very small amount of 
it. makes a sharp instrument seem dull. 

Method. — The skin of the ear or finger and the sticker 
are cleaned with alcohol or ether, which is allowed to evapo- 

1 Bass. C. C. " A practical, inexpensive, aseptic blood-sticker. ' ' Med. 
Record, 1910. LXXVIII. 53 S. 

226 



THE BLOOD 



227 



rate completely. The skin is then pierced. The point of 
the sticker should be held close to the skin and pushed in 
rather quickly ; beginners frequently make a sudden stab at 
the part from a distance of several inches, either missing 
the skin entirely or producing an unnecessarily deep wound. 
The wound must be such that the blood flows freely from it ; 
squeezing the tissues to obtain blood is not permissible, 
since the blood is thereby diluted with lymph. 



COUNTING THE BLOOD CORPUSCLES 

The Hemocytometer.— In blood counting the standard 
instrument in universal use is the hemocytometer of Thoma 




0.100mm. 
4b<J mTn 


II C.Zeiss 
lljgHl Jena. 







Fig. 39, 



-The Thoma-Zeiss Hemocytometer. a, the counting chamber; b, 
same in section; S, the diluting pipette. 



the 



(Fig. 39). The instrument as originally designed by 
Thoma was not satisfactory for the enumeration of the 
leukocytes, and, as a result of this, numerous modifications 
of the Thoma ruling of the counting chamber have been 
brought forward. 1 The change consists in an increase of 

1 The writer learns from dealers in laboratory supplies that they are forced 
to keep the original counting chamber in stock, since physicians specify 
"Thoma-Zeiss" in ordering. This is doubtless due to the fact that the pur- 
chasers are unfamiliar with the much more practical rulings — modifications of 
the Thoma — which are mentioned above. 



228 



COUNTING THE BLOOD CORPUSCLES 



the ruled area from 1 sq. mm. to 9 sq. mm. Neubauer, 
Turk, Zappert-Ewing, and others have devised rulings, 
which are designated by their names. The writer prefers 
the Neubauer ruling (Fig. 40). In all the modifications 
the central square millimeter retains the original ruling of 

Thoma. The addi- 
tional eight square 
millimeters sur- 
round it, and are 
solely for greater 
accuracy and con- 
venience in counting 
the white cor- 
puscles. 

The Eulikg of 
the Counting 
Chambek. — T here 
is, then, a ruled area 
3 mm. on a side or 
9 sq. mm. (Fig. 40). 
This area is divided 
into nine large squares, each of which is 1 mm. on a side 
or 1 sq. mm. The central square millimeter, which is used 
for counting the erythrocytes, is subdivided into 400 small 
squares, each of which is -^ mm. on a side, and has, there- 
fore, an area of^^-sq. mm. 1 By means of double lines 
these smallest squares are grouped into blocks of twenty- 
five, a convenient unit to employ in counting. The ruling 
of the remaining eight large squares (1 sq. mm. each) varies 
according to the design selected. For the leukocyte count 
the entire nine square millimeters may be used. 

1 This is always indicated on the glass slide of the counting chamber — 
^qmm. ,; The depth is also given— " Tiefe 0.100 mm." 




Fig. 40. 



-The Neubauer Ruling of the Hemo- 
cytometer. 



THE BLOOD 229 

CONSTRUCTION OF THE COUNTING ChAMBEK (Fig. 39). 

The ruling is on a glass disc (B) which is mounted on a 
heavy glass slide (o). The disc is surrounded by a glass 
table (W), also attached to the slide, the surface of which 
is exactly one-tenth (0.1) of a millimeter above that of the 
ruled disc. A moat (r) about 2 mm. wide separates the 
disc from the table. When the cover glass (D), which is 
supplied with the apparatus, is placed upon the glass table, 
it thus forms a space between its under surface and the 
surface of the ruled disc, which is 0.1 mm. deep. 

The Diluting Pipettes. — Since whole blood is much too 
thick to permit direct enumeration of its corpuscular ele- 
ments, pipettes with which accurate dilutions of the blood 
can be made are required. Two pipettes are furnished 
with the complete hemocytometer, one for the red cells, the 
other for the white. Each consists (Fig. 39) of a capillary 
tube, which opens into a bulb containing a glass pearl. 
The capillary tube is divided into 10 equal parts. In the 
red pipette the bulb, when filled to the line on its upper out- 
let (marked 101), holds one hundred times the contents of 
the ten divisions of the capillary tube. It is, therefore, 
possible to obtain ten different dilutions of blood, if desired. 
Practically, only two dilutions are employed — 1:200 and 
1:100. 

With the white pipette lower dilutions are made. The 
bulb usually contains either ten or twenty times the con- 
tent of the capillary tube. Dilutions of 1 :10, 1 :20, etc., are 
generally made. 

Procedure in Counting the Erythrocytes 

(1) Diluting Fluids.— The requirement for the dilut- 
ing fluid is that it preserves well the red corpuscles. Nu- 



230 COUNTING THE BLOOD CORPUSCLES 

merous formulae have been elaborated. Among the better 
known of these are the following: 

(a) Hayevn's solution: 

Bichlorid of mercury 0.5 gm. 

Sodium chlorid 1.0 gm. 

Sodium sulphate 5.0 gm. 

Distilled water 200.0 c. c. 

Dissolve and preserve in a tightly stop- 
pered bottle. 

This is the most satisfactory diluting fluid. It keeps 
indefinitely, and no organisms grow in it. The red cells 
settle evenly in it. 

(b) Physiological salt solution : 

Sodium chlorid 0.85 gm. 

Distilled water 100.0 c.c. 

Dissolve. 

The red cells settle slowly and often unevenly in salt 
solution. 

(c) Toisson's fluid: 

Sodium sulphate 8.0 gm. 

Sodium chlorid 1.0 gm. 

Glycerin (neutral) 30.0 c. c. 

Distilled water 160.0 c. c. 

Methyl violet q. s. 

Dissolve. (The methyl violet is added in 
minute amount (25 to 30 mg.), just enough to 
color the fluid, which should remain clear and 
transparent. ) 



THE BLOOD 231 

Toisson's fluid is not stable and must be filtered before 
using. Low forms of vegetable life luxuriate in it, and it 
is, on the whole, an unsatisfactory fluid, its only advantage 
— which is usually negligible — being that leukocytic and 
other nuclei are stained. 

(2) Filling the Pipette.— The first essential is to have 
the blood flowing freely and to obtain a fresh drop. If it 
is necessary to squeeze the ear to obtain the blood, the 
latter will be diluted with tissue lymph, making the count 
too low; if the drop is not perfectly fresh, clotting will 
have begun, so that a uniform suspension of the cells can- 
not be secured. The blood is sucked cautiously into the 
capillary tube to the line marked 0.5. (With anemias of 
2,500,000 cells or less, the blood is more conveniently drawn 
up to the mark 1 to secure a lower dilution, i. e., 1:100.) 
If the blood is accidentally sucked above the line, it may be 
lowered by drawing the finger across the tip of the pipette, 
provided the column of blood has not passed more than 1 
mm. above the line; if it has extended farther, the blood 
adhering to the wall of the tube will be sufficient to intro- 
duce a serious error in the dilution. In the latter case the 
blood should be drawn up to the next line on the capillary 
tube, 0.6, quickly, and a corresponding correction in dilu- 
tion calculated. Bubbles of air in the column of blood 
must, of course, be avoided. After the tube has been ac- 
curately filled with blood, the end of the pipette is wiped 
free of blood, and is then plunged into the diluting fluid, 
which is sucked up to the line marked 101. While the 
diluting fluid is being drawn up, the pipette, held between 
thumb and fingers, is revolved to keep the glass pearl with- 
in the bulb in motion, both for the purpose of mixing the 
blood and diluting fluid and also to prevent bubbles adher- 
ing to the pearl, which would render the dilution inaccu- 



232 COUNTING THE BLOOD CORPUSCLES ' 

rate. The filling of the capillary tube and the subsequent 
dilution of the blood require rapid manipulation to prevent 
clotting. When the fluid reaches the line 101, the mouth- 
piece of the pipette is occluded with the tongue. The finger 
is then jDlaced over the tip of the capillary tube, the thumb 
grasping the other end of the pipette. An even and uni- 
form suspension of the erythrocytes is now secured by 
shaking the pipette, held horizontally, for at least two min- 
utes. After the shaking is completed, several drops are 
blown out of the pipette to thoroughly empty the capillary 
tube (which contained only diluting fluid), and the count- 
ing chamber is then filled. Since the red corpuscles settle 
quite rapidly, the contents of the pipette must be perfectly 
mixed by shaking each time before filling the counting 
chamber. 

(3) Filling the Counting Chamber.— The counting 
chamber and cover glass must be perfectly clean and free 
from dust. A drop of the diluted blood is placed at one 
side of the ruled disc; the cover glass, used as a lever, is 
gradually lowered onto the drop, the edge of the glass table 
serving as the fulcrum. As the cover glass comes in con- 
tact with the drop, and the latter spreads over the surface 
of the ruled disc, there will be a tendency for one or more 
bubbles to form. By alternately raising and lowering the 
cover glass the fluid will spread evenly, and bubbles can be 
avoided. The size of the drop of diluted blood which is 
taken is important, and is learned by practice. It should 
be just large enough to cover the surface of the ruled disc, 
when the cover glass is applied. If it is so large that 
much of the fluid runs into the moat or that any fluid is 
found between the cover glass and glass table, the counting 
chamber must be cleaned and refilled. Before proceeding 
to count the erythrocytes, two conditions must be fulfilled 



THE BLOOD 233 

in addition to those already given: (1) Newton's rings 
(prismatic colors) must be visible between the cover glass 
and glass table when looked at obliquely toward the light, 
since this proves that the two surfaces are in close apposi- 
tion and are not separated by a particle of dust, which 
would deepen the chamber; and (2) inspection under low 
magnification should show no clots and no gross inequali- 
ties in the distribution of the red cells over the ruled sur- 
face. These conditions being fulfilled, after allowing the 
erythrocytes to settle in the fluid two or three minutes so 
that all of them will be in the same focus, the enumeration 
of the cells is proceeded with. 

In making the count it is advisable, if the worker is 
inexperienced, to use a high magnification (Leitz ocular 1, 
objective 6, or corresponding values for other makes of 
microscope), which will include a single block of twenty- 
five small squares. Ordinarily, however, a lower magnifica- 
tion is sufficient, such, for example, as one obtains with 
Leitz ocular 3, objective 3, with the tube drawn out to its 
full length. 

(4) The enumeration of the cells may be made in an al- 
most endless number of ways. The method described by 
Emerson 1 has been employed by the writer, and is as fol- 
lows : The cells in 200 small squares are counted. This is 
accomplished by counting separately eight blocks of twen- 
ty-five small squares, the blocks used being those at the 
corners of the ruled area from each of two preparations. 
A little more time is consumed in refilling the counting 
chamber, but it serves as an additional check on the accu- 
racy of one's technique. Beginning at the upper left cor- 
ner of a block of twenty-five small squares, the count is 
made from left to right in the upper tier of five small 

1 Emerson, C. P. ''Clinical Diagnosis." 



234 COUNTING THE BLOOD CORPUSCLES 

squares, then from right to left in the second tier, and so 
on, nntil the entire block of twenty-five has been covered. 
The cells touching the line on two adjacent sides of a square 
are counted, while those on the line of the two remaining 
sides are disregarded. The total count for each block of 
twenty-five small squares is set down. When all eight 
blocks have been counted, the difference between the high- 
est and lowest total count should not exceed 25 cells. If 
this limit is exceeded, the distribution of the cells in the 
counting chamber has been so uneven as to make the re- 
sults untrustworthy. 1 

(5) Calculation* of the Result.— The calculation is sim- 
ple. Since each small square is -fa mm. on a side, and 
the counting chamber is -fa mm. deep, the cubic content of 
one small square is i x - 1 x -i- or 1 • c. mm. As 

H 20 20 10 4000 

200 such squares have been counted, it follows that the 
sum of all the cells counted is the number of cells contained 
in -2o_Q or i c. mm. of diluted blood. Since the dilution 

40 2 

used was 1 :200, the total number of cells counted must be 
multiplied by 20 and by 200, or by 4,000, to obtain the num- 
ber of cells in 1 c. mm. of undiluted blood, the desired re- 
sult. 

The normal count usually given for healthy adults is: 
for males, 5,000,000 ; for females, 4,500,000 cells per c. mm. 
These figures represent averages. The red count of nor- 
mal adults is not infrequently as high as 6,000,000. 

(6) Cleaning the Apparatus.— (a) The Counting Cham- 
ber. — The counting chamber should be cleaned with water 
only. After thorough rinsing, it is wiped dry with a soft, 
clean cloth. The diluted blood should never be permitted 
to dry in the counting chamber. Alcohol, ether, or similar 

1 Following Emerson, students are required to fulfill these conditions and 
to make counts on two successive days, which shall not differ by more than 
200,000 cells per c. mm., the limit of error of the method. 



THE BLOOD 235 

solvent should not be employed, as it may dissolve the 
cement, by which the ruled disc and the glass table sur- 
rounding it are fastened to the slide. (In case this acci- 
dent happens, all the parts may be returned to the maker, 
for it is possible at times to repair the damage.) 

(b) The Pipettes. — The pipettes should be cleaned im- 
mediately on completion of the count. If this is impossible, 
they should at least be emptied and refilled with water, un- 
til it is convenient to clean them. If allowed to stand, the 
diluted blood drying in the tip of the capillary may occlude 
it. The successive steps are as follows: 

(1) The pipette is emptied of its contents. 

(2) Distilled water or clear tap water is drawn into 
the pipette. After emptying, fill it with — 

(3) Ethyl alcohol, 95 per cent. Shake the pipette so 
that the water adhering to the pearl is mixed with the al- 
cohol, place the rubber tubing over the tip of the capil- 
lary tube, and blow the alcohol through. 

(4) Fill the pipette with ether, and shake again. Re- 
move the rubber tubing, and allow the ether to run out by 
inverting the pipette. 

(5) Aspirate air through the pipette till the walls of 
the bulb are dry and the glass bead rolls freely as the pi- 
pette is rotated. If the bead sticks to the wall, it means 
that there is still moisture remaining, or that the pipette 
is not clean. 

• A suction pump is a great convenience in cleaning the 
pipettes. As a substitute for the pump, a stiff -walled rub- 
ber bulb may be employed. 

If blood clots in the capillary tube, it should be removed 
as soon as possible by means of a horse hair. For this 
purpose hairs from the tail or mane are washed in water 
and alcohol and kept in the latter. Wire should never be 



236 COUNTING THE BLOOD CORPUSCLES 

used, as it may scratch the glass, and there is also great 
danger of chipping the end of the pipette. At times, if 
the clot has become very firm or dry, it is necessary to 
place the pipette in a test tube with nitric acid. When a 
film of coagulated albumin forms over the inner surface 
of the bulb, the pipette may be filled with nitric acid and 
set aside for several hours. 

Counting the Leukocytes 

(1) Diluting Fluid.— For counting the white cells of the 
blood it is necessary to have a fluid which will render the 
erythrocytes invisible and cause the leukocytic nuclei to 
stand out prominently. Dilute acetic acid is universally 
employed for this purpose. It is used in about 1 per cent, 
strength. The solution is quickly prepared by adding two 
drops of glacial acetic acid to 10 c. c. of distilled water. 
The dilute acid should be prepared freshly each day; yeast 
cells grow in it, if it is kept, and may lead to confusion, 
since single cells resemble the nuclei of lymphocytes, while 
several cells are not very unlike a polymorphous nucleus. 
It is convenient to have a small, wide-mouthed bottle with 
a file-scratch indicating 10 c. c. for preparing the dilute 
acetic acid. 

(2) Filling the Pipette.— The capillary tube of the white 
pipette is larger in caliber than that of the red pipette, 
and, therefore, a larger drop of blood will be required to 
fill it. The blood is sucked up to the mark 0.5, as a rule; 
the blood on the end of the pipette is wiped off, and the 
tip immediately plunged into the diluting fluid, which is 
sucked up to the mark 11 (or 21 in the case of the larger 
white pipettes). The pipette must be rotated more jerkily 
and the fluid sucked in more slowly than with the red 



THE BLOOD 237 

pipette, to avoid the air bubble which so often clings to the 
glass pearl. After the pipette is filled the end is occluded, 
and the pipette, held horizontally, is shaken at least two 
minutes. In short, all the precautions requisite to a proper 
filling of the red pipette (to which the reader is referred) 
apply with equal force here. Bubbles in the column of 
blood or in the bulb of the pipette ruin the preparation. 
The column of blood must be drawn quickly and accurately 
to the desired mark. 

When the number of leukocytes is greatly increased, as 
is the case in extreme leukocytoses and usually in leukemia, 
it is often more convenient to use the red pipette for the 
leukocyte count in order to obtain a greater dilution. 

(3) Filling the Counting- Chamber.— The counting 
chamber is filled in the manner described under the red 
cell count. Since the capillary tube of the white pipette is 
wider, the diluted blood flows out of it more rapidly, and 
the size of the drop is less easily controlled. A drop of the 
right size, Newton's rings, and an even distribution of the 
cells over the ruled area are essential. 

The magnification employed should be the same as that 
used in counting the erythrocytes (q. v.). Work is much 
more rapid with the lower power. 

(4) The Enumeration of the Leukocytes.— After the 
cells have settled until all are in the same focus (usually 
in two or three minutes), the leukocytes, whose nuclei stand 
out as refractive bodies, are counted in one square milli- 
meter at a time, including the cells which touch the line 
on two sides of the square only. Nine or ten square milli- 
meters are counted — nine in one preparation, or five in 
each of two preparations. The procedure is the same as 
that used in counting the red corpuscles, the only difference) 
being the larger unit employed, 1 sq. mm. The difference 

17 



238 COUNTING THE BLOOD CORPUSCLES 

between maximal and minimal total count for 1 sq. mm. 
should not exceed 8 cells. 

The normal leukocyte count of adults lies between 5,000 
and 10,000 cells per c. mm. — rarely 12,000 cells. 

If the diluting fluid is not freshly prepared, yeast cells 
may be counted and lead to serious error. A second source 
of error is the presence of a considerable number of nu- 
cleated red cells in the blood. Ordinarily erythroblasts 
are detected only when the stained blood is simultaneously 
examined ; and, as they are numerous only in marked path- 
ological states of the blood, they seldom escape notice. 
They cannot well be separated from the leukocytes in the 
counting chamber, so that the count obtained is the sum 
total of all nucleated cells in the blood — both red and white. 
The number of erythroblasts is determined by making a 
differential count of the stained blood and noting the rela- 
tive number of nucleated red cells as compared with the 
leukocytes. From the proportion of the two kinds of cells 
found, the correction of the white count is made. Thus, if 
the leukocyte count were 20,000, and differential count 
showed 125 nucleated reds to 500 leukocytes, the relative 
frequency of the two would be as 1:4. Therefore, there 
are present in one cubic millimeter of blood 5,000 nucleated 
red cells. Since these were included with the leukocytes 
in the total count, the latter must be corrected by deducting 
the nucleated reds. The leukocyte count thus becomes 
15,000 instead of 20,000. 

(5) Calculation of the Result.— Let us assume that the 
dilution employed was 1 :20, and that nine square milli- 
meters were counted. Each square millimeter has a cubic 
content of 0.1 c. mm., since the counting chamber is 0.1 mm. 
deep. The sum of the leukocytes in the nine large squares 
divided by 9 gives the average number of cells in 0.1 c. mm. 



THE BLOOD 239 

of diluted blood. To obtain the number of cells in 1 c. mm. 
of undiluted blood, this number is multiplied by 10 and by 
20 (dilution 1:20), or by 200. 

Biirker' s Modification 1 of the Thoma Counting Cham- 
ber.— By substituting two wedge-shaped, ruled glass plates 
for the ruled disc, Biirker has modified Thoma 's design. 



^^^^^^^^HHBxAaKfi^t^ *£' 


P^ U 




1 iHhjE^H 


^^^HIRhiP^^' 



Fig. 41. — Burker's Hemocytometer. (Zeiss.) 

The wedges are placed with their bases opposite one 
another (Fig. 41), the apices extending out toward the 
sides of the glass slide. Two oblong tables at either side 
of the wedges with long axes running parallel replace the 
glass table which surrounds the ruled disc in the Thoma 
pattern. The cover glass, when resting on the tables, 2 is 
0.1 mm. above the ruled surface of the wedges, as in the 
Thoma apparatus. The ruling is after a special design of 
Biirker. 3 Each wedge is ruled, and the chamber has, there- 

1 Biirker, K. ' ' Erf ahrungen mit den neuen Zaklkammer nebst einer wei- 
teren Verbesserung derselben. " Arch. f. d. ges. Physiol., 1907, CXVIII, 460. 

2 When ordered, clips are supplied with the apparatus to hold the cover 
glass firmly in place. 

3 By special order Zeiss makes the Biirker counting chamber with Neu- 
bauer ruling. 



240 COUNTING THE BLOOD CORPUSCLES 

fore, two ruled areas in place of the one of the Thoma 
chamber. 

In filling the Biirker hemocytometer for counting, the 
first step consists in placing the. cover glass on the tables, 
Newton's rings being obtained. Then the blood is allowed 
to run under the cover from the pipette by capillarity. 
One side may be filled for the red count, the other for the 
white. 

Biirker calls attention to the following technical points 
which should be observed: (a) To avoid bubbles, the cover 
glass and chamber must be carefully cleaned. Further- 
more, in using pipettes having an angle at their tips, bub- 
bles are prevented with difficulty ; the tip should be rounded 
off with emery paper, (b) The drop of blood which flows 
under the cover glass should not be so large as to overflow 
into the gutter. Counting chambers should not be accepted 
in which the gutter between the two ruled surfaces is less 
than 2 mm. wide and the lateral gutters 1.5 mm. wide, (c) 
After allowing the red cells to settle for at least three min- 
utes, the evenness of distribution should be determined be- 
fore proceeding with the count. Biirker advises that this 
be accomplished in the following manner: The counting 
chamber is placed on the stage of the microscope, illumi- 
nated by the mirror with the diaphragm opened wide. By 
viewing the counting surface obliquely with the unaided 
eye one sees a film or veil formed by the erythrocytes. Ir- 
regularities in the distribution of the cells are shown by 
variations in the density of the film. When such irregu- 
larities are visible, the chamber must be refilled. In count- 
ing the leukocytes, this procedure is not applicable, for the 
cells are too few. Microscopic examination with low power 
must be made. 



THE BLOOD 241 



Counting the Eosinophilic Leukocytes 

The number of eosinophilic cells is usually arrived at 
by making a total count of the leukocytes in the ordinary 
way and, at the same time, preparing stained specimens. 
By finding the percentage of eosinophiles in a differential 
count, the absolute number of cells per cubic millimeter 
may then be calculated. 

Dunger 1 has devised a method by which the absolute 
number of eosinophiles per c. mm. may be determined di- 
rectly. 

Dunger's Method.— The formula of the diluting fluid is: 

1 per cent, aqueous solution of eosin. . 10.0 c. c. 

Acetone 10.0 c. c. 

Distilled water 90.0 c. c. 

The solution must be preserved in a tightly corked bottle 
to prevent evaporation of the acetone, and is then quite 
stable. 

A 1:10 dilution of the blood is made in the white pi- 
pette, and the mixture is thoroughly shaken three to five 
minutes. After blowing out the contents of the capillary 
tube, a drop is placed in the counting chamber (ruled for 
leukocyte count, i. e., 9 sq. mm.). Only the eosinophile 
cells are well seen ; they appear as small, pink bodies. With 
a magnification of 120 to 150 diameters they are readily 
seen. The entire nine square millimeters of the chamber 
are counted. Ordinarily 9 to 18 eosinophile cells are found 
in this area; this corresponds to about 100 to 200 eosino- 

1 Dunger, E. ' ' Eine einf ache Methode der Zahlvmg der eosinophilen 
Leukocyten imd der praktische Wert dieser Untersuehung. ' ' Milnchen. med. 
Wchnschr., 1910, LVII, 1942. 



242 COUNTING THE BLOOD CORPUSCLES 

philic leukocytes per c. mm. The calculation of the total 
number of cells is made in the way described for counting 
the leukocytes (p. 238). After a little practice an increase 
in the number of these cells is recognized at a glance. 
By making a leukocyte count in the usual manner simul- 
taneously, the percentage of eosinophiles may be deter- 
mined. 

Counting the Blood Platelets 

Several methods have been proposed for counting the 
platelets. The indirect method has given fair results, i. e., 
making a count of the erythrocytes in the usual way and 
at the same time determining the relative number of plate- 
lets as compared to the red cells in a fresh specimen of 
blood. The number of platelets is then calculated. Direct 
methods of counting platelets have been attempted; the 
only one which appears to give reliable results is that of 
Wright and Kinnicutt. 

Method of Wright and Kinnicutt. 1 — The diluting fluid: 

Solution 1: 

"Brilliant cresyl blue" 1.0 gm. 

Distilled water 300.0 c. c. 

Dissolve. Keep on ice to prevent the 
growth of yeasts. 

Solution 2 : 

Potassium cyanid 1.0 gm. 

Distilled water 1,400.0 c. c. 

Method. — ' ' The blood is mixed with the diluting fluid in 
the proportion of 1:100 by means of the pipette used for 

1 Wright, J. H., and Kinnicutt, E. "A new method of counting the blood 
platelets for clinical purposes and some of the results obtained with it." 
Jour. A. M. A., 1911, LVI, 1457. 



THE BLOOD 243 

counting red blood corpuscles, and the counting is done in 
the ordinary counting chamber with a high power dry ob- 
jective. In order to render the platelets more clearly vis- 
ible, the specially thin cover glass of Zeiss, with central 
excavation, is used (cover glass No. 146, Zeiss catalog 1 ). 
The diluting fluid consists of two parts of the aqueous solu- 
tion of 'brilliant cresyl blue' (solution 1), and three parts 
of the aqueous solution of potassium cyanid (solution 2). 
These two solutions must be kept in separate bottles and 
mixed and filtered immediately before using. Of course, 
the pipette should be well shaken after withdrawing the 
sample for counting. After the counting chamber is filled, 
it is left at rest for ten or fifteen minutes, in order that 
the blood platelets may settle to the bottom of the cham- 
ber and be more easily and accurately counted. 

"The platelets appear as sharply outlined, round or 
oval or elongated, lilac-colored bodies, some of which form 
a part of small spheres or globules of hyalin, unstained 
substance. 

"The red cells are decolorized and appear only as * shad- 
ows,' so that they do not obscure the platelets. The nuclei 
of the white cells are stained a dark blue, the protoplasm 
light blue. If the technique is correct, there should be no 
precipitate in the preparation. 

"The cresyl blue solution is permanent, but should be 
kept on ice in order to prevent the growth of yeasts. The 
cyanid solution should be made up at least every ten days. 
It is, of course, necessary that the solution be made from 
pure potassium cyanid, which has not undergone decompo- 
sition. As already stated, the two solutions must be mixed 
and filtered immediately before using, because after filtra- 

1 This special cover glass is, however, unnecessary, if one has the usual 
thin cover glass, which permits the use of the high power dry objective. 



214 HEMOGLOBIN DETERMINATIONS 

tion, if the mixture is allowed to stand exposed to the air 
for a short time, a precipitate will form in it. After the 
diluting fluid has been mixed with the blood in the pipette. 
however, no precipitate forms, and. as the platelets do not 
quickly break up in the mixture, the counting may be done 
after some hours, if necessary. For example, a count im- 
mediately after filling the pipette was 258,000 and another 
count from the same filling of the pipette made eighteen 
hours later was 253,000. 

"A proper technique yields a remarkably even distribu- 
tion of the platelets in the chamber. For all practical 
purposes, the counting of the platelets in 100 small squares 
is sufficient, but for greater accuracy all 400 small squares 
should be counted, or 200 small squares in each of two 
fillings of the chamber." 

With their method Wright and Kinnicutt find that the 
platelet count of normal adults varies between 226,000 and 
367,000 per cubic millimeter, the general average being 
297,000. 

HEMOGLOBIN DETERMINATIONS 

Many methods for the determination of the amount or 
percentage of hemoglobin have been brought forward. For 
a description of all of them the reader is referred to the 
textbooks of hematology. 

(1) Tallqvist devised a color scale, which has been 
widely used. It consists of a series of ten shades of red. 
intended to represent the color intensity of hemoglobin 
from 10 per cent, to 100 per cent. Each color is perforated. 
A drop of blood is collected on a filter paper, supplied in 
the book containing the scale, and, as soon as the gloss 
has disappeared from the drop, it is placed under the per- 
foration in one of the red strips. It is moved until the 



THE BLOOD 245 

color of the drop of blood corresponds with one of the 
shades of red. This represents the hemoglobin percentage 
of the blood. With the Tallqvist scale it is possible, per- 
haps, to make a more accurate guess as to the percentage 
of hemoglobin than without it. It is well recognized that 
the scale is very inexact. In fact, the color scales in sep- 
arate books do not always match. When the blood is 
hydremic, the plasma runs beyond the corpuscles, which 
are concentrated at the center of the drop, introducing an 
additional error in the very cases where more exact results 
are desirable. If a hemoglobin determination is indicated 
a little more time should be spent than is required with 
the Tallqvist scale, in order to obtain a result of some 
value. 

(2) Sahli's Hemometer.— Sahli's hemometer is a modi- 
fication of the old Grower instrument. It consists (Fig. 42) 
of one tube containing the standard solution and a second 
tube of the same caliber graduated from to 140, each divi- 
sion representing 20 c. mm. The tubes are placed in a 
hard rubber stand, which has an opaque glass back. A pi- 
pette with a line representing 20 c. mm. is supplied with 
the instrument. The standard solution is one of acid hema- 
tin, prepared as follows: 1 

Blood 1 part 

~q hydrochloric acid 10 parts 

Distilled water to 50 parts 

Mix and add — 
Glycerin 50 parts 

The hemoglobin is converted into acid hematin, which 

1 Hastings, T. W. "The estimation of hemoglobin-content of blood with 
modern instruments. ' ' Jour. A. M. A., 1907, XL VIII, 1749. 



246 HEMOGLOBIN DETERMINATIONS 

does not go into solution, but is in a very fine state of sus- 
pension. Therefore, the hematin settles slowly, when the 
instrument is not in use, and for this reason a glass pearl 
is placed in the tube to facilitate mixing the standard 
fluid, which should be done each time immediately before 
using. Sahli ' obtains the blood for the standard solution 




Fig. 42. — The Sahli Hemometeh. 

from young adult males having a high red cell count. This 
explains the fact that normal blood seldom shows more 
than 90 per cent, of hemoglobin with the Sahli hemometer, 
when a new instrument is employed. In the course of 
time the standard solution fades. If the tube is protected 
from the light when the instrument is not in use, however, 
it may be kept as long as two years or more without se- 
rious deterioration. It is well to check the standard solu- 

1 Sahli, H. "Diagnostic Methods." 1st Amer. Ed., Phila. & London, 
1905, p. 620. 



THE BLOOD 247 

tion from time to time with several bloods of normal adults 
having 5,000,000 red cells. With such a count the hemo- 
globin percentage should be 100. If the reading of the 
hemometer is too high or too low, the percentage of error 
is noted and the readings are then corrected correspond- 
ingly. 

It is more satisfactory to prepare the standard solution 
from blood with a 5,000,000 count, checking it with other 
similar bloods. By doing this the standard tube may be 
refilled, say, every six or twelve months, doing away with 
the necessity of corrections. The values obtained are then 
safe for use in determination of the color index. 

It is very important that the standard tube and the 
graduated tube have the same diameter. 1 If unequal, 
it is clear that the results obtained will be without 
value. 

Method. — The graduated tube is filled accurately to the 
mark 10 with tenth normal hydrochloric acid. The pipette 
is now filled with blood exactly to the line marked 20 c. mm. 
The blood is quickly discharged into the acid in the grad- 
uated tube, and the pipette is rinsed two or three times 
with the acid to remove that which adheres to the wall of 
the pipette. The graduated tube is immediately shaken 
to secure a uniform suspension of the blood before clotting 
will have begun. The blood quickly becomes dark brown 
in color from the conversion of the hemoglobin into acid 
hematin. The mixture of blood and acid is allowed to 
stand exactly one minute, and is then diluted with water, 
until its color matches that of the standard solution. Day- 

1 Tubes from uniform tubing have been made for several years for the 
writer by Eimer and Amend, Third Ave., New York City. The standard tube 
is made in the form of the usual test tube. When filled with the standard 
solution it may be sealed in the flame, though it is more convenient to use a 
paraffined cork, as in this way it may be refilled an indefinite number of times. 



248 HEMOGLOBIN DETERMINATIONS 

light or artificial light may be used, since the tubes contain 
the same substance. 

When the graduated tube is inverted to secure thorough 
mixing, care should be exercised that none of the fluid ad- 
heres to the finger, for enough may be removed in this way 
to cause a considerable lowering of the reading. When 
comparing the colors, it is well to rotate the graduated 
tube until the lines on it are not visible. When the colors 
have been accurately matched, the instrument is set aside 
for a couple of minutes to allow the fluid in the graduated 
tube adhering to the wall to run down. The height of the 
column is then read. This gives the hemoglobin percen- 
tage, the color of the standard fluid being considered as 
100 per cent. 

In cases where the hemoglobin is extremely low it is 
difficult to obtain satisfactory readings. In such case 40 
c. mm. of blood may be added to the acid. The final result 
is then divided by 2. 

Staubli 1 has made a critical study of this method of 
determining hemoglobin, using the Sahli hemometer and 
the Autenrieth-Konigsberger colorimeter. He finds that 
there is a progressive darkening of the acid hematin formed 
by mixing the blood with the tenth normal acid. The dark- 
ening is most rapid in the first few minutes after the mix- 
ture is prepared; plotting the values obtained, he found 
that the curve is a parabola. He has demonstrated that 
it is important to use tenth normal hydrochloric acid, not 
an approximate dilution, and to measure it into the grad- 
uated tube accurately, for the rapidity of darkening of the 
blood is directly proportional to the quantity and concen- 

1 Staubli, C. ' ' Zur Ausf iihruiig der Hamoglobinbestrrrmmng. (Unter 
Umwandlung des Hamoglobins in salzsaures Haniatin.) " Miinchen. med. 
Wclinsclir., 1911, LVUI, 2129. 



THE BLOOD 249 

tration of acid. The blood-acid mixture 'should be allowed 
to stand exactly one minute, as Sahli recommends, and 
should then be quickly diluted with water, which inhibits 
the effect of the acid. Staubli suggests that a better method 
of procedure with the Sahli hemometer is as follows : The 
blood-acid mixture is diluted at once with tenth normal 
hydrochloric acid, until the color is approximately that of 
the standard tube ; then wait for ten minutes 1 to make the 
final comparison. The final dilution, which will require 
only a few drops, may be made either with water or with 
the acid. This technique in his hands has yielded uniform 
results with all bloods. 

"Whichever method is followed, it is absolutely essential 
that it be adhered to strictly in order to obtain comparable 
results. 

Aside from variations in the standard fluids and pos- 
sible lack of uniformity in diameter of the tubes, it is prob- 
able that Staubli 's findings explain to a great extent the 
anomalous results which many workers have obtained with 
this instrument. 

Cleaning the Hemometer. — The graduated tube is rinsed 
with water. The pipette is rinsed first with water, then 
with alcohol and with ether. Finally, air is aspirated 
through it to dry the pipette. 

(3) The Fleischl-Miescher Hemoglobinometer.— This 
instrument is generally considered to be the most accurate 
for the determination of hemoglobin. It is not well adapted 
to general use, since it is expensive and requires a dark 
room for making the readings. 

The instrument (Fig. 43) consists of a standard, on 
which a wedge of red glass is mounted, cells of 12 and 15 

1 Ten minutes is the time interval selected, since it was found that the 
darkening which occurs beyond this interval is slight. 



250 



HEMOGLOBIN DETERMINATIONS 



mm. depth, and a mixing pipette. The pipette is similar 
to those used with the hemocytometer. The markings on 
its capillary tube, y 2 , Y^ and 1, permit of dilutions of the 
blood of 1 :400, 1 :300, and 1 :200 respectively. Sodium car- 
bonate, 0.1 per cent, aqueous solution, is used as the dilut- 
ing -fluid. The cells, 12 and 15 mm. deep respectively, are 




Fig. 43. — The Fleischl-Miescher Hemoglobinometer. 

divided into two equal parts, one of which is filled with 
water, the other with the diluted blood. Each compart- 
ment should be filled until the surface of the fluid is con- 
vex above the upper level of the cell. The cell is then 
sealed by a glass disc, care being exercised to avoid bubbles 
in the fluids. Finally, a metal cap is placed over the glass 
disc. In the cap there is a slit, which should be so placed 
that its long axis is at a right angle to the partition divid- 
ing the cell. The cell is now placed on the stand, so that 
the compartment containing water is directly above the red 



THE BLOOD 251 

glass wedge. Candle light furnishes the most satisfactory 
illumination; it must be used in a dark room. All direct 
rays of light are cut off from the eye of the examiner, 
either with a large cone or by placing the instrument in a 
box with one side — at which the operator stands — open and 
a small hole cut in the opposite side near the bottom for 
illumination of the reflector. The colored prism is now 
moved until the shades of red in the two divisions of the 
cell are alike. The reading is made on the scale and re- 
corded. Ten such readings should be made, and the aver- 
age of them taken. If the cell 15 mm. deep has been used, 
the glass disc is removed and the diluted blood sucked 
back into the pipette. The 12-mm. cell is then filled, and 
ten readings are made with it, and the average taken. The 
latter should be four-fifths of the reading obtained with 
the 15-mm. cell, and should not vary by more than 2 per 
cent. The readings obtained do not represent hemoglobin 
percentages. They are to be used in connection with the 
table found in a pamphlet supplied with each instrument. 
(As the instruments are separately standardized, the tables 
often differ, and, therefore, cannot be used interchange- 
ably.) From the table the hemoglobin in grams per 100 
c. c. of blood is calculated according to the directions in the 
pamphlet. All values are to be reduced to a dilution of 
1:300 with the 15-mm. cell. (For normal blood the 1:300 
dilution is usually employed. With anemic blood use a 
dilution of 1:200, and with plethoric 1:400.) 

The normal hemoglobin value with the Fleischl-Miesch- 
er apparatus is subject to considerable variation. Emer- 
son 1 finds that in normal young adults the mean hemo- 
globin per 1,000,000 cells is 2.63 gm. 

In leukemia or with extreme leukocytoses readings may 

1 Emerson, C. P. "Clinical Diagnosis." 1st Ed., pp. 466 et seq. 



252 HEMOGLOBIN DETERMINATIONS 

be difficult, because of the opacity produced by the white 
cells. The leukocytes may be removed by centrifngalizing 
the diluted blood before filling the cell. 

Cleaning the HemogJobhwmeter. — The pipette is cleaned 
in the same manner as the counting pipettes (q. v.). The 
cells should be taken apart, washed with water, dried, and 
reassembled. 

(4) Haldane's Hemoglobinometer.— Haldane's hemo- 
globinometer is a very satisfactory instrument. Its only 
drawback — a minor one — is that illuminating gas is re- 
quired in its use. Like the Sahli hemometer. it is a modi- 
fication of the original Gower apparatus. 

(5) Dare's Hemoglobinometer.— Dare's hemoglobino- 
meter gives excellent results, but is fragile and expensive. 

Sulphhemoglobinemia, 1 Methemoglobinemia.— The rec- 
ognition of these abnormal pigments in the blood is de- 
scribed as follows by Clarke and Curts: 1 The blood is 
drawn from a vein. or. if this is not allowed, a few drops 
from the finger or ear will usually suffice. It is immediately 
diluted with twice its volume of distilled water, before 
clotting has taken place, and is thoroughly shaken. After 
the fibrin has separated, the solution is filtered several 
times through one filter paper, and the clear solution looked 
at through the spectroscope. The solution is then diluted 
drop by drop with water, until the red color | of the spec- 
trum) stands out clearly. If there is a black absorption 
band in the red, either methemoglobin or sulphhemoglobin 
is present. If such a band persists after the addition of a 
drop of dilute ammonium sulphid. the pigment is sulph- 
hemoglobin; if it disappears, it is methemoglobin. 

In the blood the two bands of oxyhemoglobin are always 

1 Clarke, T. W., and Curts. E. M. "Sulphhemoglobinemia. with a report 
of the first case in America."' Med. Eecord, 1910, LXXVIII. 987. 



THE BLOOD 253 

visible. In addition to these sulphhemoglobin presents a 
band in the red (near the orange) midway between C and 
D. With methemoglobin the band is again in the red, but 
nearer to C. (Compare with Fig. 7.) 

COLOR INDEX 

The color index is the quotient obtained by dividing 
the percentage of hemoglobin by the percentage of red 
corpuscles, 5,000,000 cells per 1 c. mm. being considered as 
100 per cent, of corpuscles. Normally, the color index is 
about 1. When the index is less than 1 it indicates that 
the average corpuscle is poor in coloring matter, whereas 
with a high index the corpuscles are abnormally rich in 
hemoglobin. 

VOLUME INDEX 

The volume index of the blood was first studied by 
Capps. 1 He introduced the term to designate the quotient 
of the percentage volume of the erythrocytes divided by 
the percentage number of these cells. 

Method. — To determine the volume of the red corpuscles, 
the hematokrit is employed. The usual form of apparatus is 
a hand or electric centrifuge armed with a frame for carry- 
ing two capillary tubes. The tubes are graduated from 
to 100. Of the various procedures which have been pro- 
posed, Capps recommends the following: The capillary 
tube is completely filled with blood, the distal end of the 
tube smeared with vaselin, and placed in the carrier of 
the hematokrit. "Two conditions are essential to prevent 
coagulation, viz., scrupulous cleanliness of the tubes and 
speed in operation. The latter condition requires that the 

1 Capps, J. A. "A study of volume index. Observations upon the volume 
of erythrocytes in various disease conditions." Jour. Med. Research, 1903, X, 

367. 

18 



254 VOLUME INDEX 

blood must be placed in the heniatokrit within a few sec- 
onds of withdrawal. ... It is desirable always to fill 
two tubes as a control of one's results. The machine should 
be operated for three minutes at a uniform speed of ten 
thousand revolutions a minute" (Capps). The tubes are 
now examined, and it is seen that the corpuscles have been 
thrown to the distal end, leaving the clear serum proxi- 
mally. With normal blood and a count of 5,000,000, the 
red corpuscles extend to the line marked 50, occupying one T 
half the capillary tube. This is the normal, and represents 
100 per cent, volume. The erythrocytes are counted at the 
same time that the volume determination is made. The 
volume index = volu l me P er cent » 5,000,000 corpuscles being 

number per cent., 

considered 100 per cent. In normal blood the volume in- 
dex is 1. Owing to variations in the size of the erythro- 
cytes in anemias, the percentage volume does not run par- 
allel to the percentage number, as a rule. 1 The volume 
index, then, expresses the relative volume of the average 
red cell as compared with the normal. 

In determining the volume of the red corpuscles, the 
leukocytes separate as a paler, grayish layer above the 
erythrocytes. Where their number is greatly increased, 
as in leukemia, determination of the volume of the red 
corpuscles is impossible. 

The hematokrit furnishes a ready means of making 
macroscopic examination of the blood serum. Lipemia, 
cholemia, and hemoglobinemia may be revealed in this man- 
ner, if sufficiently marked, though hemoglobinemia may be 
an artefact from mechanical injury to the red corpuscles. 

Cleaning the Hematokrit Tubes. — Blood should be 

1 Capps (Joe. cit.) reports extremely interesting observations on volume 
index compared -with, color index and with measurements of the erythrocytes in 
primary and secondary anemias. 



THE BLOOD 255 

blown out of the tubes as soon as the reading has been 
made. The tubes are cleaned by drawing water, alcohol, 
and ether through them successively. If they are not per- 
fectly cleaned, use acetic acid first, then the other fluids 
in the order given. 

MEASURING THE DIAMETER OF CELLS 

The diameter or a dimension of microscopic objects 
is, expressed in micra (designated by the Greek letter /x), 
one micron being the thousandth part of a millimeter (0.001 
mm.). In making measurements an ocular micrometer is 
employed. This is a glass disc, on which fifty equal divi- 
sions are marked by parallel lines. The upper lens of the 
eye-piece is unscrewed, and the micrometer is inserted in 
the tube of the ocular. 1 The value of the divisions on the 
micrometer scale is now determined in the following man- 
ner: The magnification of the microscope is varied by 
three factors, namely, the objective, the ocular, and the 
tube length. The usual tube length employed is 160 mm. 
Using this, the value of the spaces on the micrometer is 
determined with the objective and ocular to be used by 
comparison with an object of known dimensions. The most 
convenient object for this purpose is the counting chamber. 
The ruled area is placed under the microscope, and the 
number of divisions of the micrometer scale, which fall 
between the opposite sides of one of the smallest squares, 
or, in the case of low magnifications, between the sides of 
a larger unit, is found. Knowing the dimensions of the 
ruled surface, it is a simple calculation to compute the 

1 In ordering an ocular micrometer, the name of the maker of the micro- 
scope should be given, as the micrometer of one make may not fit the ocular 
of another. 



256 VISCOSITY OF BLOOD AND OTHER FLUIDS 

value of a single division of the micrometer scale. The 
smallest squares are ^V mm - on a s i^ e ? or 50 micra. 

As applied to the blood, measurements are usually made 
on stained films. At least one hundred cells should be 
measured, and, where much anisocytosis exists, two hun- 
dred cells should be the minimal number. 

In the measurement of oval bodies, such as the eggs of 
many parasites, the two dimensions are readily obtained by 
rotating the ocular through ninety degrees. 

VISCOSITY OF THE BLOOD AND OTHER FLUIDS 

For clinical use, a number of instruments for deter- 
mining viscosity have been described. That of Hess 1 has 
proved very satisfactory. It is compact and easily por- 
table. The determinations may be made with a little prac- 
tice in two or three minutes. The subject of viscosity is 
well discussed by Austrian. 2 The viscosity is compared 
with that of water. 

Method of Hess. — The Hess viscosimeter (Fig. 44) con- 
sists of an opaque glass plate (H) on which two gradu- 
ated tubes, A and B, are mounted. At one end these tubes 
communicate with a T-tube, G, which in turn is connected 
by rubber tubing with the rubber bulb L. At the other end 
the graduated tubes connect with capillaries C and D. The 
latter open into tubes E and F, which have the same diam- 
eter as the graduated tubes A and B. Capillary tube C and 
tube E are made in one piece, while tube F is held in ap- 
position with tube D by means of the clip X. It is remov- 
able, and a number of similar tubes are supplied with the 

1 Hess, W. ' ' Ein neuer Apparat zur Bestimmung der Viskositat des 
Blutes." Munchen. med. Wchnschr., 1907, LIT, 1590. 

2 Austrian, C. E. " The viscosity of the blood in health and disease. ' ' 
Johns Hopkins Eosp. Bull., 1911, XXII, 9. 



THE BLOOD 237 

instrument. Through the valve Q it is possible to shut off 
the communication of the graduated tube B with the T- 
tube, and, therefore, with the rubber bulb as well. Between 
the rubber bulb and the rubber tubing a short piece of 
glass tubing is inserted; in it a hole is blown. This is 
opened or closed with the finger, and permits instant re- 
lease of the negative pressure produced by the suction of 
the bulb. A thermometer is mounted on the glass plate. 
Method. — With a pipette, which is furnished with the 



fiU.Ut.JLUi v ' - L ^ . 


it . * 


c ? 




T^ : iff" 










N 
M 



L 
Fig. 44. — The Viscosimeter of Hess. (After Austrian.) 

instrument, distilled water is placed at the opening of tube 
E. The valve Q is opened, and by suction from the bulb 
L the water is drawn into the tube E, until it reaches the 
capillary tube C. The pipette is then withdrawn, and the 
column of water is sucked further, until it reaches the mark 
on the scale of the graduated tube A. The valve Q is 
then closed, the pressure having been released by remov- 
ing the finger from the opening. (It is unnecessary to 
refill the tubes A, C, E, with distilled water for each de- 
termination; the water may be allowed to remain in the 
tubes and used repeatedly.) The tube F is then touched 
to a fresh drop of the blood to be examined. The blood 
should enter at the pointed end of the tube. When the lat- 
ter is about three-fourths full the tube is held so that the 
blood will run down to the funnel-shaped end of the tube, 
which is then placed in contact with the free end of the 



258 VISCOSITY OF BLOOD AND OTHER FLUIDS 

capillary tube D. and held in position by the clip X. By 
suction with the bulb the column of blood is then drawn to 
the line on the scale of the graduated tube B. when the 
pressure is again released. The valve Q is now opened, 
and by suction through the bulb the column of blood is 
drawn to the mark 1 on the scale. It is drawn exactly to 
the mark, when the pressure is removed by withdrawing 
the finger from the opening. The point on the scale to 
which the water has been drawn represents the degree of 
viscosity of the blood. The viscosity of the blood of nor- 
mal adults is about 4.55 'Austrian). If the viscosity of 
the blood is very great, or if the blood coagulates rapidly. 
the column of blood is drawn to the mark % or 1 2- an ^ the 
result obtained is multiplied by 4 or 2 respectively. The 
error arising from making the observations at ordinary 
room temperatures is negligible (Austrian). 

Cleaning the Viscosimeter. — As soon as the reading is 
made, positive pressure is exerted to expel the fluids from 
the graduated tubes. When the water reaches the zero 
line the valve Q is closed. The tube F is removed and the 
blood which escapes from the capillary tube D is caught on 
filter paper or cloth. A second tube, filled with concen- 
trated ammonium hydrate, is placed in the clip, and am- 
monia is drawn through the tubes A. I). at least 2 cm. be- 
yond the line 1 to which the blood extended. The ammonia 
is expelled and the tubes are refilled with fresh ammonia, 
which is allowed to remain in the tubes until the instru- 
ment is used again. The end of the capillary tube D is 
closed with a rubber cap. Immediately before using the 
apparatus the cap is removed and the ammonia expelled. 
If the pressure used to expel the ammonia is slight, only 
a trace remains, which is without appreciable effect on 
the result. It is essential that the tubes be perfectly clean. 



THE BLOOD 259 

If the apparatus is unused for some time, difficulty may be 
experienced in forcing the ammonia out of the tubes. This 
is usually due to the formation of ammonium salts at the 
opening of the capillary tube; they may be removed by 
solution in water. The valve should be lubricated with 
vaselin. The tubes F, after use, may be cleaned by as- 
pirating water through them and then placing them in 
nitric acid for several hours. They are then dried by suc- 
cessive rinsings with water, alcohol, and ether. Erroneous 
results may be obtained if the tubes are dirty. The instru- 
ment should be tested from time to time with distilled 
water. If the result is not 1, the tubes are to be cleaned by 
drawing nitric acid into them. After an hour or so the 
acid is removed, the tubes rinsed twice with water, and 
then with ammonia. 

THE SPECIFIC GRAVITY OF THE BLOOD 

In clinical work the method usually used for determina- 
tion of the specific gravity of the blood is that of Hammer- 
schlag. A mixture of chloroform (sp. gr. 1.485) and ben- 
zol (sp. gr. 0.88) is placed in a cylinder. The specific grav- 
ity of the mixture should approximate that of normal blood 
(1.050 to 1.062). A capillary tube is filled with blood, which 
is flowing freely from the puncture wound, and a drop is 
allowed to fall into the mixture. If the drop sinks, its 
specific gravity is greater than that of the mixture, and 
more chloroform is added; if the reverse holds good, ben- 
zol is added. After each addition of chloroform or benzol 
the contents of the cylinder are well stirred. When a mix- 
ture is finally obtained in which the drop of blood neither 
sinks nor rises, its specific gravity is determined with an 
areometer. The result is approximately the specific grav- 
ity of the blood. 



260 THE SPECIFIC GRAVITY OF THE BLOOD 

As Xaegeli suggests, a series of mixtures of varying 
specific gravity is a great convenience. 

The blood should not be permitted to drop into the 
chloroform-benzol mixture from a height, as it scatters. 
A bubble in the drop of blood may lead to serious error. 
Quick work is necessary to prevent the extraction of much 
water from the blood, and also to avoid evaporation of the 
mixture. After use the mixture may be filtered and kept 
in a brown glass bottle. 

The specific gravity of blood plasma or serum may be 
determined by the method of Hammerschlag. Xorinally it 
lies between 1.029 and 1.032. 

For the more accurate and time-consuming methods of 
determining specific gravity the reader is referred to works 
on hematology. 

THE COAGULATION TIME OF THE BLOOD 

The methods of determining the coagulation time of 
the blood are many, and the results obtained with each 
are more or less divergent. Xo perfectly satisfactory 
method has been brought forward. Among the best is 
that which employs the apparatus of Broclie and Eussell, 
as modified and inrproved by Boggs. 1 Eesults almost 
as uniform have been published by Hinman and Sladen, 2 
using their modification of Alilian's method. An essen- 
tial prerequisite to any method is absolute cleanliness 
of the apparatus and of the skin at the site of punc- 
ture. 

1 Boggs, T. E. "Some clinical aspects of blood coagulation." Internet. 
Clinics, 1908, I (18th series), 31. 

2 Hinman, F., and Sladen, F. J. " Measurement of the coagulation time 
of the blood and its application." Johns Hopkins Hosp. Bull., 1907, XVIII, 
207. 



THE BLOOD 261 

(1) The Method of Brodie and Russell, as Modified by 
Boggs.— The instrument (Fig. 45) consists of a moist 
chamber A and a truncated glass cone B, mounted to fit 
into the former. The lower surface of the cone is 4 mm. 
in diameter. Through the wall of the chamber a metal 
tube C extends, the tip of which approximates the lower 
surface of the cone. By means of a rubber bulb, such as 
is used on a camera, attached to the outer end of the metal 
tube, a current of air may D E 

be directed tangentially to jj '\ j^\\ ' 

the lower surface of the r^ 



cone. The upper surface of r 1 — 

the cone is covered by a — 

cover glass D-E; at E there ,» , K ^ , ., 

& ' riG. 45. — Boggs Modification of the 

is a pinhole. Coagulometer of Brodie and 

,, , , -j . -. n Russell. A, moist chamber; B, cone 

M et li o a. — A drop ot of glass the lower surface Q f which 
blood is placed on the lower holds the dr °p of blood ; c - side tube ' 

connecting with bulb; D and E, cover 
end Of the glaSS COne, and glass; at E, a pinhole. (After Emer- 

the cone inserted in the son) 

chamber, which is then placed on the stage of the micro- 
scope. The drop is examined with the low power, and at 
the same time the bulb is squeezed, directing an air current 
against the periphery of the drop of blood. At first the 
corpuscles move freely in a circular direction. As clotting 
begins, masses of corpuscles take the place of the single 
cells. As coagulation progresses, the masses of corpuscles 
tend to become fixed in the drop. The air now displaces the 
masses in the direction of the current, but they spring back 
immediately after the air ceases to disturb them. The next 
stage, which is taken as the end-point, differs from the pre- 
ceding in that a very gentle blast of air produces "radial 
elastic motion, as of a rubber ball pressed in at one point 
and released" (Fig. 46). When this point is reached the 



262 THE COAGULATION TIME OF THE BLOOD 

time is again taken. The cone is immediately removed and 
the blood is wiped off on a cloth or filter paper to confirm 
the existence of a clot. 

Occasionally a drop of blood fails to clot at one point. 
If this happens the result is valueless, and a second deter- 
mination must be made. 

The normal coagulation time of the blood with this in- 
strument is between three and eight minutes, usually about 
G.ve minutes (Hinman and Sladen). 

A /? 





Fig. 46. — Diagram to Illustrate the Movement of the Cells during 
Coagulation. D, the end-point. (After Emerson.) 

Boggs emphasizes the following points of technique: 
The blood must be flowing freely from the puncture. When 
a sufficiently large drop has collected, the cone is touched 
to it at right angles to the surface of the drop and not 
dipped in it. In this way a drop of constant size is ob- 
tained. The coagulation time begins with the appearance 
of the drop of blood in the wound. Pressure upon the tis- 
sues and congestion of the parts are to be avoided, as they 
tend to increase the coagulability of the blood. Absolute 
cleanliness of the apparatus is essential. The air current 
should be gentle, and should not be applied too frequently. 



THE BLOOD 263 

(2) Milian's Method, as Modified by Hinman and Sla- 

den. 1 — This method is extremely simple, requiring only 
clean glass slides and a millimeter scale. The ear is punc- 
tured, the first drop is discarded, and the time counted 
from the first appearance of the second drop. The lobule 
of the ear is held out, and the under-surface of the slide 
touched to the drop of blood, so that several small drops 
are obtained on it. The slide is then turned quickly to 
prevent the drops from flowing. Placing the slide over a 
scale, only' those drops having diameters of 4 and 5 mm. 
are allowed to remain, others being wiped off. There are 
two methods of watching the drops to determine when 
coagulation has occurred; in each the slide is held verti- 
cally. In the one the profile of the drop is observed; be- 
fore coagulation the drops sag, assuming the shape of a 
tear, while the uniform convexity is preserved after co- 
agulation is complete. In the other method the vertical 
slide is examined by transmitted light. The denser por- 
tion will be found about the center of the drop, when coagu- 
lation has occurred; while the blood is still fluid the de- 
pendent part of the drop is the denser. The presence of a 
clot is then confirmed by transferring the drop to a cloth 
or filter paper. 

Compared with Boggs' method, in which a 4-mm. cone 
is employed, the authors find that a 4-mm. drop clots more 
rapidly, a 5-mm. drop more slowly. They therefore take 
the mean coagulation time of several drops of 4 and 5 mm. 
diameter, respectively. The majority of records fall be- 
tween five and eight minutes. Below eight minutes a rec- 
ord is within the limits for a normal coagulation time. 

1 Hinman, F., and Sladen, F. J. "Measurement of the coagulation time 
of the "blood and its application. ' ' Johns Hopkins Hosp. Bull., 1907, XVIII, 
207. 



264 RESISTANCE OF BED BLOOD CORPUSCLES 

Anything above eight minutes is delayed. TThen the co- 
agulation time is delayed, only 5-mm. drops are used, in 
order to minimize the error due to evaporation. 

THE RESISTANCE OF THE RED BLOOD CORPUSCLES 

Numerous substances have been employed, against 
which the resistance of the red blood corpuscles has been 
measured. Solutions of sodium chlorid of varying strength, 
notably hypotonic solutions, have been most extensively 
used, and with them results of clinical importance have 
been obtained 

Method. 1 — Under aseptic precautions 2 to 5 c. c. of 
blood are aspirated from an arm vein, and immediately 
placed in live to ten times the volume of 1 per cent, sodium 
fluoric! or 1.5 per cent, sodium citrate in 0.85 per cent, so- 
dium chlorid to prevent clotting. As soon as the blood is 
discharged into the fluid, the flask is shaken well to insure 
thorough mixture. The blood-fluorid mixture is now cen- 
trifugalized at high speed to throw down the corpuscles. 
The supernatant fluid, containing the greater part of the 
blood plasma, is poured off. The plasma is then com- 
pletely removed by washing the corpuscles three times in 
0.85 per cent, solution of sodium chlorid. After the last 
washing the supernatant fluid is pipetted off. leaving the 
erythrocytes at the bottom of the centrifuge tube. 

The hypotonic solutions of sodium chlorid diminish 
from 0.85 per cent, by 0.03 per cent., the solutions being 
M . v 2 per cent.. 0.79 per cent., and so on. down to 0.25 per 
cent. They are quickly prepared by filling one 50-c. c. 
burette, graduated to -L c. c. with distilled water, and 

L 1 r : =5. W. L. -'Paroxysmal hemoglobinuria: Blood studies in three 
cases.'* Johns Hoplins Hosp. Bull, 1911. XXII. 23 S. 



THE BLOOD 265 

another with 1 per cent, aqueous solution of sodium chlo- 
rid. Thus, to prepare 10 c. c. of 0.70 per cent, sodium 
chlorid, take 7 c. c. of the 1 per cent, salt solution and 3 
c. c. of distilled water. 

A series of small test tubes is appropriately marked 
and placed in a rack. To each there are added 3 c. c. of 
hypotonic salt solution and 0.03 c. c. (about one drop) of 
the red blood corpuscles. The salt solution and blood cor- 
puscles are well mixed by shaking. (The tubes are, of 
course, perfectly clean and sterile, and are plugged with 
cotton.) After all have been filled, they are placed in the 
ice chest to prevent bacterial growth, and are allowed to 
remain until the red cells have settled to the bottom. For 
the lower dilutions this usually requires about two hours. 
The supernatant fluid is now examined for free hemoglobin, 
the presence of which shows that there has been laking of 
the corpuscles. 

The tube of lowest dilution showing even a trace of 
hemoglobin in the fluid represents the so-called minimal 
resistance. That is, with this strength of salt solution the 
least resistant cells are "laked," their hemoglobin escap- 
ing from the cell membrane into the salt solution. The 
maximal resistance is found by noting the strength of salt 
solution in which all the red corpuscles are laked. 

Normally the minimal resistance, in terms of hypotonic 
salt solution, is about 0.47, the maximal resistance about 
0.30. 

THE EXAMINATION OF FRESH AND STAINED PREPA- 
RATIONS OF BLOOD 

The first requisite in the preparation of fresh or dried 
films of blood is perfectly clean glassware. 

The Cleaning of Cover Glasses and Slides.— Of the vari- 



266 FRESH AND STAINED PREPARATIONS OF BLOOD 

ous methods used To clean glassware for Wood work in the 
author's laboratory, the following has given the most satis- 
factory results, and is always dependable: 

1 1 ) Immerse the covers or slides | in concentrated sul- 
phuric acid for about twenty-four hours. 

_ Pour off the acid and wash in running water. 

( 3 i Drain off the water and cover the glassware with 
95 per cent, alcohol for an hour or longer. 

(4) Replace the alcohol with chloroform and dry the 
glassware as needed. 

The covers should be dried with a perfectly clean cloth, 
free from lint. An old linen handkerchief which has been 
laundered many times is suitable. If the glassware is to 
be kept dry. it should be placed in a dust-proof receptacle. 

Ether may be substituted for chloroform, but is less 
S£ tisfactory. 

For blood work n 4 -in. square cover glasses. Xo. 1. are 
the best. The 3xl-in. glass slides should be thin, with 
straight, even eds:es. if they are to be employed in making 
blood films. If cover glasses are used in making the films, 
the finish of the slide is less important. 

ExAvnxATiox of the Feesh Blood 

In the examination of the fresh blood, a procedure 
which is too generally neglected, the specimen is prepared 
in the following manner : The ear is pierced, but the punc- 
ture should not be so dee] as t use a very free now of 
blood, since it is essential to be able to regulate the size 
of the drop accurately. Therefore, a small, superficial 
puncture is made, from which the blood will escape easily 
on i tl pressure. (Pressure is to be avoided as far 

as possible, to prevent the dilution of the blood with tis- 



THE BLOOD 267 

sue lymph. In grasping the ear the fingers should be at 
least y 2 inch from the wound. It is better, of course, to 
obtain the blood without any pressure whatever.) A drop 
of blood about the size of a small pinhead is transferred 
to a cover slip, which is immediately placed upon a glass 
slide. The blood spreads out between the cover glass and 
slide in a thin film. Microscopic examination should show 
the individual red corpuscles separated from one another 
in the central portion of the film, with the thicker parts 
at the periphery presenting rouleaux formation. If the 
cells are not separated the drop of blood used was too large, 
provided the glassware was clean and the drop of blood 
fresh. 

Failure to obtain satisfactory specimens is usually at- 
tributable to one of several causes. If the drop of blood is 
allowed to remain on the ear an appreciable length of time 
before it is used, clotting may have begun ; this, of course, 
interferes with the proper spreading of the blood. Again, 
any dirt on the ear also has the same effect. Particles of 
dust or bits of lint on the glassware prevent even uniform 
spreading of the blood by elevating the cover glass from 
the slide. As dust frequently settles on the cover glasses 
or slides while preparing to secure the blood, it is a good 
plan to remove all such particles by blowing on the glass 
(avoid moisture from the breath on the glass), or by brush- 
ing the surfaces with a camel's hair brush. Any grease 
or dirt of any kind on the glass makes it impossible to 
obtain good specimens. It is advisable to handle the cover 
glasses with a pair of straight forceps, to avoid the grease, 
etc., of the fingers. 

Sealing the Fresh Specimen.— If the specimen is to be 
kept for any length of time, it should be sealed to prevent 
drying. Vaselin is convenient for this purpose. A small 



268 FRESH AND STAINED PREPARATIONS OF BLOOD 

quantity of it is taken np on the end of a match, which is 
then rapidly passed through a Bunsen flame. The edge 
of the cover glass is now lined with the melted vaselin, 
which hardens almost instantaneously, and effectually seals 
the specimen. Paraffin of low melting point may also be 
used. Specimens prepared in this way may be kept for 
a surprising length of time with little alteration in the red 
corpuscles. 

The Preparation of Dry (Permanent) Blood Smears.— 
(1) The Cover Glass-Forceps Method. — In the writer's ex- 
perience the best results are obtained by using two cover 
glasses. The covers are cleaned and dried as described on 
page 265. Any particles of dust are carefully removed 
from the covers just prior to making the smear. Forceps 
are used to avoid soiling the surfaces of the cover glasses 
with the fingers. With care, however, perfectly satisfac- 
tory films may be made with the fingers. 

Two pairs of forceps are needed. One is a cross-billed 
forceps, which will hold a cover glass firmly. The spring 
should be strong and the blades perfectly parallel, so that 
the grip on the cover slip will be uniform. If the forceps 
are suitable it should be possible to lift them by grasping 
a cover glass caught between the blades of the forceps 
without changing the relative position of the cover glass. 
Forceps which cannot withstand this simple test usually 
prove to be useless. A pair of straight forceps is also re- 
quired. They should be fairly stiff, with blades having 
plain, square ends. When holding a cover slip firmly only 
the tips of the blades should touch it. 

To prepare blood films a clean cover glass is placed in 
the cross-billed forceps, the puncture wound is then wiped 
free of blood, and, when a drop of the proper size appears 
(about the size of a small pinhead with a normal count, 



THE BLOOD 269 

larger with anemic blood), it is taken up on a second cover 
slip held in the straight forceps. This is immediately 
placed on the first cover glass. The blood spreads out be- 
tween the two in a thin layer. Just before the drop will 
have stopped spreading between the covers, the overlap- 
ping edge of the second cover is grasped with the straight 
forceps, and the two are quickly pulled apart. It requires 
considerable practice to pull the covers apart in exactly 
parallel planes, which is necessary if the spreads are to 
be good. With good preparations microscopic examination 
will show the individual red cells well separated over one- 
half to two-thirds of the preparation. With a little ex- 
perience good smears may be selected with the unaided 
eye. When inspected by transmitted light, the area in 
which the cells are properly separated resembles an ex- 
tremely thin, gray veil; if the cells are grouped in little 
islands, the uniformity of the veil is lost. The thick parts 
of the smear are more dense and opaque. 

The films, which are allowed to dry in the air, are then 
ready for fixing and staining. At times, when the humidity 
is very high, it may be necessary to fan the films to hasten 
the drying. (During the fly season films should be pro- 
tected from the pests, as they may eat practically all the 
blood from a cover glass in a few seconds.) 

The size of the drop of blood is a matter of great im- 
portance in making blood smears with the cover glass 
method, as has been indicated above. The correct size 
will depend largely on the number of red corpuscles in the 
blood. With very anemic patients, whose blood is thin 
and hydremic, a relatively large drop will be needed. The 
general tendency of beginners is to take a drop which is 
too large. If this mistake is made, no part of the film 
is thinly spread, the erythrocytes being piled up so that 

19 



270 FRESH AND STAINED PREPARATIONS OF BLOOD 

study of the individual cells is impossible. If one waits 
until the blood has stopped spreading, it is often impos- 
sible to separate the covers, as they become sealed. Lint, 
dust, gritty particles, or grease on the cover glasses will 
make it impossible to secure satisfactory specimens. 

(2) The Glass Slide Method. — Many clinicians prefer 
glass slides to cover glasses in making blood films. 1 The 
method requires practically no practice, and is simpler 
than the cover glass-forceps method. The area of the blood 
film may be made much larger than that obtainable on a 
cover glass. The slides should be thin, and should have 
perfectly smooth, even edges and level surfaces. They 
must, as a matter of course, be perfectly clean. Any dust 
which may have settled on the slides should be removed 
before using them. 

A drop of blood considerably larger than that required 
in the cover glass method is taken up on the end of one 
slide, which is then approximated to the surface of a sec- 
ond slide, placed on a table or other firm surface. The 
first slide is held at an angle of about 45 degrees to the 
second. The blood spreads out along the end of the first 
slide, which is now pushed rather rapidly along the sur- 
face of the second slide. The blood spreads out in a thin 
layer over the surface of the second slide. In making the 
spread, pressure on the slides is unnecessary. 

1 In the experience of the author, more satisfactory specimens are ob- 
tained with the cover glass method. As a rule, the leukocytes are more evenly 
distributed over the specimen. The large smear, which is obtained with the 
slide, is usually no advantage, for it is seldom the case that a greater area is 
needed than is contained in a cover glass preparation. The great value of the 
slide method, aside from the fact that good smears may be obtained with it, 
lies in the fact that the technique is easily acquired, and fair specimens may 
often be obtained with slides which have been cleaned only with water. All 
laboratory workers should, therefore, be able to employ the method, though the 
cover glass method is preferred. 



THE BLOOD 271 

Labeling the Blood Films. — With specimens made on 
glass slides, where the area of the blood film is large, a 
part of it may be employed for labeling the specimen. A 
very simple and practical method has been described by 
von Ezdorf. 1 The necessary data is written on the thick 
part of the film with a soft, black lead pencil. The label 
thus made is permanent, and is not affected by staining or 
washing the specimen. The black contrasts well with the 
usual pink color of the film. 

Fixation of Blood Smears.— Various methods are avail- 
able for fixing the blood cells to the slide. The following 
will be found useful : 

(1) Heat Fixation. — A triangular copper bar, first in- 
troduced into blood work by Ehrlich, is usually used for 
heat fixation. The bar is placed on a tripod with a Bun- 
sen flame under the tip of the bar. In a short time, if pro- 
tected from strong drafts, all parts of the bar acquire and 
maintain a fairly constant temperature. By dropping 
water from a pipette onto the bar, the point farthest from 
the flame is determined, at which the drop of water remains 
spheroidal and rolls off. The temperature at this point, 
the " spheroidal point" for water, is about 150° C. The 
point is marked, and the blood films, with the specimen 
side up, are then placed just inside this point, i. e., toward 
the flame from the spheroidal point, and allowed to remain 
30 to 45 seconds. This usually suffices to fix the films well. 
In certain instances a longer or shorter time is required, the 
extremes falling between 5 and 120 seconds. By placing 
four specimens of blood (cover glass preparations) at the 
spheroidal point and removing them at the end of 30, 35, 
40, and 45 seconds respectively, and staining all, the proper 

1 Von Ezdorf, R. H. "The labeling of dried blood films." Jour. 
A. M. A., 1910, LIV, 125. 



272 FRESH AND STAINED PREPARATIONS OF BLOOD 

fixation time is quickly determined with the great majority 
of bloods. It is to be remembered that there is no one 
optimal fixation time applicable to all bloods. A separate 
determination must be made for each individual blood ex- 
amined. 

In place of the copper bar, an oven may be used. The 
specimens are placed in the oven, which is maintained at 
a temperature ranging between 110° and 120° C. for one 
to two hours, rarely longer. By removing and staining a 
specimen every fifteen minutes after the first hour, the 
correct fixation time is determined. This method of em- 
ploying heat fixation is particularly convenient, when a 
large number of specimens of the same blood are to be 
fixed. 

Heat fixation is always used with Ehrlich's triacid 
stain. In using Pappenheim's methyl green-pyronin mix- 
ture, heat fixation is also to be preferred. It is less use- 
ful for other blood stains. 

(2) Ethyl Alcohol. — The specimens may be fixed by im- 
mersion in absolute alcohol one to five minutes, or in 96 per 
cent, alcohol five to twenty minutes. They are then dried 
in the air or between blotting paper. Less expensive and 
about as satisfactory is the denatured alcohol of com- 
merce. 

This method of fixation is useful in connection with 
hematoxylin and eosin, methylene blue, etc. 

(3) Methyl Alcohol. — Absolute methyl alcohol, acting 
for one to five minutes, is an excellent fixative. (Methyl 
alcohol fixation is carried out as a part of the staining tech- 
nique in employing Leishman's, Wilson's, and Jenner's 
stains, as will appear below.) 

Methyl alcohol may be used in place of ethyl alcohol, 
and is usually employed with Giemsa 's stain. 



THE BLOOD 273 

(4) Acetone. — The specimens are placed in acetone five 
minutes, and are then dried in the air. 

(5) Alcohol-F ormalin. — Futcher and Lazear have used 
0.25 per cent, formalin in 95 per cent, alcohol. The solu- 
tion must be prepared freshly, and is obtained by adding 
one drop of commercial formalin (40 per cent.) to 10 c. c. 
of alcohol. The specimen is allowed to remain in this mix- 
ture one minute, and is then washed in water and blotted 
dry. 

This method is the best for staining with carbol-thionin. 

Staining the Blood 

The stains and combinations of stain for blood work 
are numerous. Those described below are among the most 
serviceable, and enable one to make all routine examina- 
tions. Most of the stains are applied to dried, fixed films 
of blood. The staining of the fresh, unfixed blood, the so- 
called " vital" staining of the blood, forms an exception. 

"Vital" Staining of the Blood 

Various stains and methods have been proposed for 
vital staining of the blood. Practically any basic dye may 
be used for this purpose, but the stains which have been 
used most extensively are Unna's polychrome methylene 
blue (Griibler's), Pappenheim's methyl green-pyronin, and 
neutral red. Only the first is described, since the picture 
obtained is rather more brilliant. 

(1) Vaughan's 1 Method.— A small puncture is made 
in the ear, and over the wound, from which the blood has 
been wiped, a minute drop of Unna's polychrome blue is 

1 Vaughan, V. C, Jr. ' ' On the appearance and significance of certain 
granules in the erythrocytes of man." Jour. Med. Eesearch, 1903-4, X, 342. 



274 FRESH AND STAINED PREPARATIONS OF BLOOD 

placed by means of a clean glass rod. A small drop of 
blood is now pressed out of the wound, so that it flows 
directly into the stain. The procedure is now the same as 
in the preparation of a specimen of fresh blood (q. v.). 
The relative proportions of stain and blood are quickly 
learned by experiment. There should be more blood than 
stain in the mixture. After the specimen has spread out 
between cover glass and slide, it is ready for examination. 
It should be sealed, if the examination is to be a prolonged 
one. 

On microscopic examination with the oil immersion ob- 
jective, the majority of the red corpuscles appear quite 
like those in a preparation of fresh blood, except where 
the stain is concentrated ; here the cells may show a diffuse 
purplish tint of varying intensity. Laking may occur in a 
certain number of the corpuscles. In some of the red cells 
granules stained bluish-purple are seen. These basophilic 
substances have been designated "granulo-reticulo-filamen- 
tous" by Sabrazes. 1 There may be very few granules in 
a cell, or they may be extremely numerous. Often the gran- 
ules appear to be attached to a delicate filament, which 
may form a part of a reticulum in the corpuscle. Not in- 
frequently the granules are clustered at the center of the 
cell, suggesting by their position and number the remnants 
of a nucleus. In erythroblasts the nucleus takes a purple 
color, as do nuclear particles, when present. The chroma- 
tin of the blood platelets takes on a similar color, and may 
often be seen occupying a position at the periphery of a 
clear, unstained globule, as Vaughan observed. Leukocy- 
tic nuclei stain more or less intensely, depending largely 

1 Sabrazes, J., and Leuret, E. l ' Hematies granuleuses et polychromato- 
philie dans 1 'ictere des nouveaux-nes. ' ' Gas. Jiebd. d. soc. med. de Bordeaux, 
1908, XXIX, 123. 



THE BLOOD 275 

on the concentration of the stain. In the protoplasm of 
the polynuclear cells, stained granules may be found. The 
ameboid leukocytes retain their activity for some time. 
The colorless cell membrane may be seen extending some 
distance beyond the granules in the pseudopods of the 
neutrophilic cells. 

(2) Method of Widal, Abrami, and Brule. 1 — " Four to 
six drops of blood are allowed to fall into a test tube con- 
taining 10 drops of a basic coloring matter, which is quite 
isotonic, and contains in addition oxalate of potassium to 
prevent the coagulation of the blood. 



Potassium oxalate, 20 per cent, solu- 
tion 2.0 c. c. 

Unna 's polychrome methylene blue . 100 drops 



La=-0.60 



"The fresh corpuscles are allowed to remain for 10 to 
20 minutes in contact with the solution, after which the 
mixture is centrifugalized, the supernatant fluid is removed, 
and the corpuscles drawn up with a pipette and placed 
upon slides, upon which they are spread as an ordinary 
drop of blood ; the covers are then dried and fixed by heat. 
Such preparations may be preserved indefinitely " (Thayer 
and Morris). 

(3) The "Dry" Method of Vital Staining.-The dry 
method of vital staining consists in spreading a thin film of 
stain on a glass slide, allowing it to dry in the air, protected 
from dust, and then placing a cover glass with a drop of 
blood on the dried stain, just as in making a prepara- 
tion of the fresh blood. The blood spreads out between 

1 Widal, F., Abrami, P., and Brule, M. "Diversite de types des 
hematies granuleuses ; pro-cedes de coloration. ' ' Compt. rend. Soc. de biol., 
Par., 1908, LXIV, 496. 



276 FRESH AND STAINED PREPARATIONS OF BLOOD 

cover and slide, the stain dissolves in the plasma, and 
the result is mneh the same as with other methods. 
With this method there is less danger of laking the cor- 
puscles. 

Pappenheim's methyl green-pyronin mixture has been 
used extensively by the French, usually with the dry 
method. 

Neutral red may be employed. A dilute solution of the 
dye is prepared in physiological salt solution, or a smaller 
quantity of saturated, aqueous solution of the stain may 
be used. It may be substituted for polychrome methylene 
blue in Yaughan's method, or may be used in the dry 
method. 

In normal blood of adults less than 1 per cent, of the 
erythrocytes contain the granulo-reticulo-filaraentous sub- 
stance, while in newborn infants the number is 7 per cent. 
or less (Vaughan). 

The Staining of Dried Blood Films 

The stains which are required for the routine examina- 
tion of blood are Ehrlich's triacid, Jenner's stain, and a 
Eomanowsky stain. For special purposes, however, other 
stains are required at times. 

(1) Methylene Blue.— (1) Fix the blood film in alcohol. 

(2) Stain with Loffler's methylene blue (p. 213) about 
3 to 5 seconds. 

(3) "Wash in water, blot dry, and mount in balsam. 

The stain is useful as a nuclear stain. For the demon- 
stration of basophilic granules and polychromatophilia, 
methylene blue is one of the most reliable stains. 

Xuclei are stained dark blue. The leukocytic granules 
are unstained, excepting basophilic granules, which take a 



THE BLOOD 277 

bluish-purple color. The basophilic protoplasm so fre- 
quently encountered in lymphocytes is stained a paler blue 
than the nucleus, the shade varying greatly in different 
cells. The erythrocytes assume a pale, greenish tint. Poly- 
chromatophilic red cells are light blue to very deep blue, 
depending on the degree of polychromatophilia. Basophilic 
granules in the red cells are stained dark blue, almost as 
dark as the nuclei. Nuclear particles in the red corpuscles 
take the same color as the nuclei of erythroblasts, i. e., a 
dark blue. Blood platelets are indistinct, appearing as 
dirty grayish-blue masses. 

(2) Eosin.— Eosin may be used as a counter-stain in 
y 2 per cent, aqueous solution. It is used after the methy- 
lene blue has been washed off the specimen. The stain is 
allowed to act a few seconds, the intensity of staining be- 
ing controlled by microscopic examination of the film in 
water. Slower staining is secured by diluting the stain- 
ing solution with water. Eosin adds little to the picture, 
except that it stains the eosinophilic granules, which now 
assume a brilliant pink or reddish-pink hue. However, 
slight polychromasia may be somewhat less evident, though 
often more striking because of the contrast. The ortho- 
chromatic erythrocytes are stained pink. If the specimen 
has been overstained with eosin, the pink color will be ap- 
parent in the protoplasm of the lymphocytes and neutro- 
philic leukocytes. 

(3) Hematoxylin.— Ehrlich's acid hematoxylin is pre- 
pared as follows : 

Solution A: 

Hematoxylin 2.0 gm. 

Alcohol, absolute 60.0 c. c. 

Dissolve. 



278 FRESH AND STAINED PREPARATIONS OF BLOOD 

Solution B : 

Saturated solution of alum in equal 
parts of glycerin and distilled 

water 60.0 c. c. 

Glacial acetic acid 3.0 c. c. 

The two solutions. A and B. are mixed and allowed to 
"ripen" in an open bottle for a week. The bottle is then 
stoppered. The ripened stain has a reddish-blue color. If 
the bottle is shaken or disturbed, the solution should be 
filtered before using. 

Method. — (a) Fix the blood films in alcohol. Heat fixa- 
tion may also be used. 

i b i Stain in hematoxylin 2 to 10 minutes or longer. 
C atrol the intensity of staining by examining the specimen 
in water. 

Wash in tap water. The washing may be com- 
pleted in a few seconds, but the beauty of the nuclear stain- 
ing is greatly enhanced by prolonged washing in tap water. 
If the specimen has been overstained with hematoxylin, it 
may be cautiously decolorized in acid alcohol | HO. 1.0 c. c. 

er cent, alcohol. 100.0 c. c. >. and again washed in water. 
It is better to avoid overstaming by controlling the stain- 
ing carefully under the microscope. 

i d ) Dry. mount in balsam. 

Hematoxylin is one of the best nuclear stains. For 
studying the morphology of nuclei it is particularly useful. 

Xuclei are stained a very dark blue, at times almost 

jk. After prolonged washing, however, the blue is 
brighter — more brilliant. As with other nuclear dyes, the 
color intensity In a given nucleus depends, of course, on 
the amount and concentration of the chromatin. Mast-cell 
granules are stained dark blue, but may be lost after wash- 



THE BLOOD 279 

ing the specimen. Other leukocytic granules are unstained. 
Basophilic protoplasm is less intensely stained than with 
methylene blue. The red blood corpuscles are lightly 
stained, and are either gray or grayish-blue. The more 
marked grades of polychromatophilia are revealed by the 
darker blue stain of the cells. Coarse basophilic granules 
in the erythrocytes are fairly well demonstrated as dark 
blue spots ; the finer granules are unstained or indistinct, 
as a rule. Nuclear particles take on a very intense, dark 
blue, like the pyknotic nuclei of normoblasts. Blood plate- 
lets are dirty blue and indistinct. 

Eosin may again be employed as a counterstain. When 
the specimen is properly stained with eosin, no pink is seen 
in the protoplasm of the neutrophilic cells, while the eosin- 
ophilic granules stand out prominently. Hematoxylin and 
eosin are useful in cases where the relative number of 
eosinophilic cells is to be determined, as a differential count 
with this point alone in view may be made rapidly. 

(4) Carbol-thionin. 

Saturated solution of thionin in 50 

per cent, alcohol 10.0 c. c. 

Carbolic acid, 1 per cent 100.0 c. c. 

(a) Fix the blood films in alcohol-formalin. 

(b) Stain with carbol-thionin Y± to 3 minutes. 

(c) Wash in water. If the specimen is overstained, the 
washing may be continued, or the specimen may be decol- 
orized in 50 per cent, alcohol. 

(d) Dry, and mount in balsam. 

The stain is an excellent nuclear stain. All nuclei are 
stained dark blue. Leukocytic granules are not specifically 
stained with the exception of the granules of the mast cells, 



280 FRESH AND STAINED PREPARATIONS OF BLOOD 

which are purple. The red blood corpuscles are greenish- 
gray. Basophilic granules are dark blue, poiychroniato- 
philic red cells varying shades of blue. Nuclei and nuclear 
particles are dark blue. The bodies of malarial parasites 
are purple, contrasting well with the red blood corpuscles. 
The nuclei of the parasites are unstained. Blood platelets 
are indistinct, and have a mauve color. 

Preparations stained with carbol-thionin fade in the 
course of several months, as a rule. 

Staining Mixtures of Two or More Stains 

(5) Ehrlich's Triacid Stain.— For the sharp differenti- 
ation of neutrophilic granules, the triacid stain of Ehrlich 
is unequaled. It should always be used in the study of 
these cells. The mixture contains three stains, two of 
which, orange G. and acid fuchsin, are acid, the third, 
methyl green 00, basic. The three basic radicals of the 
methyl green are satisfied by the acid dyes, hence the name, 
triacid. The formula * given below, a slight modification 
of the usual one, has been found to yield uniformly good 
staining mixtures. 2 whereas formerly it has been more or 
less a matter of good fortune to obtain a satisfactory solu- 
tion. Good staining mixtures may usually be had from 
Gnibler. In preparing the mixture, saturated aqueous so- 

1 Morris, E. S. "The value of Ehrlich's triacid stain in blood work." 
Jour. A. If. A.. 1910. LV. 501. 

2 Recently (1912) we have encountered the first failures in more than five 
years. After numerous experiments the cause of the trouble was found to lie 
in the acid fuchsin. Three different lots of the powdered stain in Gnibler 's 
original packages obtained through one firm resulted in poor staining mix- 
tures, while acid fuchsin secured from another firm (also Grubler "s make) gave 
very satisfactory results. With the poor acid fuchsin all cells were stained 
diffusely red. The nature of the defect in the acid fuchsin has not yet been 
determined. 



THE BLOOD 281 

lutions of methyl green, 1 orange G., and acid fuchsin are 
made separately. They must be allowed to settle for at 
least a week before use, and should be replenished as 
needed, so that a constant supply of the stock solutions 
may be on hand. Griibler's stains are generally used. 
The formula, as modified, is: 

Saturated aqueous solution of orange G 13.0 c. c. 

Saturated aqueous solution of acid fuchsin. . . 7.0 c. c. 

Distilled water 15.0 c. c. 

Absolute alcohol 15.0 c. c. 

Saturated aqueous solution of methyl green. .17.5 c. c. 

Absolute alcohol 10.0 c. c. 

Glycerin 10.0 c. c. 

The fluids are mixed with the same graduated cylinder, 
which should not be rinsed. The receiving flask should be 
shaken vigorously after the addition of each constituent, 
which is added in the order given in the formula. It is 
essential to add the methyl green, second portion of alco- 
hol, and glycerin slowly, shaking well after each addition. 
The mixture is ready for use immediately, and does not 
deteriorate with age. After the mixture has stood a while 
a small amount of precipitate may form. Care should be 
exercised that this is not disturbed when using the stain. 2 

Method of Staining. — (a) Fix the blood spread by heat 
(p. 271). 

(b) Stain 5 to 10 minutes (overstaining is impossible). 

(c) Wash quickly in water, blot dry with filter paper, 
and mount in balsam. 

In a properly fixed specimen the neutrophilic granules 

1 Methyl green is used in place of methyl green 00 of the original formula. 

2 For blood stains, bottles with droppers, the rubber nipples of which also 
serve as stoppers, are indispensable. Barnes ' bottle is a very good one. 



282 FRESH AND STAINED PREPARATIONS OF BLOOD 

stand out sharply. When this is the case the erythrocytes 
are usually, though not always, colored deep orange or 
buff; in an underfixed specimen they are stained red, while 
too prolonged fixation causes them to take a yellow color. 
The color of the red corpuscles, while a safe index of the 
fixation in most instances, fails at times. The final criterion 
by which a specimen is judged must be the staining of the 
neutrophilic granules. 

In a good specimen (plate I) the erythrocytes have, 
then, a buff color usually. Polychromasia is not demon- 
strated. Basophilic granules and Cabot's ring bodies are 
unstained. The pyknotic nuclei of normoblasts take a dark 
green color, the megaloblastic nuclei being less deeply 
stained. Often reddish areas are visible in the nuclei. Nu- 
clear particles are stained green, but are much less strik- 
ing than when stained with better nuclear stains, such as 
hematoxylin or a Eomanowsky stain. Malarial and other 
parasites are not well stained. It is evident, therefore, 
that the triacid stain is very inferior for the study of patho- 
logical changes in the red blood corpuscles. Many of the 
abnormalities of the red cells are not shown at all, and 
none of them is as well demonstrated as with a Romanow- 
sky stain or with Jenner's stain. 

Blood platelets appear as ill-defined, indefinite, mauve- 
colored masses. 

Leukocytic granules are well differentiated, but the nu- 
clei are poorly stained, assuming a rather pale green or 
bluish-green color. All neutrophilic granules take a lilac 
color. There should be no blurring of them in a well-fixed 
specimen; the granules are sharply denned and distinct, 
though in the myelocytes the very fine granules stand out 
less prominently than in the polynuclear cells. Eosinophi- 
lic granules assume a brick-red or coppery color, while 



LEGEND FOR PLATE I. 
(All drawings made with camera lucida; X 1200. Hrlich's triacic: Btaii). 

1. 2. Normal red corpuscles. 

3. )k:^:' '.: r. 

4. Normoblast. 

5. Lynipho 

6. Large mononuclear leukocyte. 

Transitional " leuko 

8. Polynuclear neutrophilic leukocyte. 

9. Polynuclear eosinophilic leukocyte. 

10. Mast cell or polynuclear basophilic leukocyte. 

11. Neutrophilic myelocyte. 

12. Eosinophilic myelocyte. 

13. Mast myelocyte or basophilic myelocyte. 

14. Myeloblast. 



PLATE I 



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n 



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THE BLOOD 283 

basophilic granules are unstained, appearing as vacuoles in 
the cytoplasm. The protoplasm of the lymphocytes is 
either colorless or a faint rose-pink. The same holds true 
for the large mononuclear and transitional cells; their 
nuclei, being poor in chromatin, usually stain very faintly, 
so that they are easily overlooked. Because of this diffi- 
culty with the non-granular leukocytes, Loftier 's methylene 
blue has been used to stain the nuclei more intensely. It is 
applied to the blood film for a few seconds (3 to 5) after 
the staining with the triacid has been completed. The 
granular stain may be slightly impaired, but the nuclei 
are much more evident. The mast-cell granules are now 
stained purple. In this connection it may be added that 
Pappenheim has prepared a triacid mixture, substituting 
methylene blue for methyl green, but it has not been widely 
adopted. 

(6) The Romano wsky Stains.— The Komanowsky stains 
are by far the best for the demonstration of pathological 
changes in the red corpuscles, and are, of course, indis- 
pensable in the study of such protozoa as the plasmodia 
of malaria, trypanosomes, etc. 

Eomanowsky's original method of preparing the stain 
has undergone numerous modifications, with a view to sim- 
plification both of the preparation of the stain and of the 
staining technique. The essential dyes are eosin and meth- 
ylene azure, the latter being obtained from methylene blue. 
Methods of preparing the stain have been described by a 
number of workers in this country, among whom may be 
mentioned Wright, Harris, Hastings, MacNeal, Wilson. 
Leishman's stain, which antedates all of those mentioned, 
is used extensively in England. The staining mixtures 
may be purchased; it is much more convenient and satis- 
factory, however, in private laboratories of physicians 

20 



284 FRESH AND STAINED PREPARATIONS OF BLOOD 

where only moderate amounts of stain are used, to buy- 
tablets of the powdered stain. A tablet is dissolved in a 
stated quantity of absolute methyl alcohol (usually 10 c. c), 
and the mixture is ready for use at once. In this way 
fresh stain may be had at frequent intervals, and there is 
less danger of deterioration of the mixture. Such tablets 
are prepared by Burroughs, Wellcome, & Co., and by Griib- 
ler. The method of preparation of only one of the modifi- 
cations of the Eomanowsky stain, Wilson's, is given. The 
writer has employed it for several years with entire satis- 
faction. 

(a) Wilson's Stain. 1 — Prepare a 1 per cent, aqueous 
solution of methylene blue, 2 which contains 0.5 per cent, 
of sodium carbonate and at least 0.5 per cent, of freshly 
precipitated silver oxid. 3 The solution is boiled for twenty 
minutes; then remove one-third of it. After boiling an- 
other twenty minutes, remove one-half. Continue to boil 
the remaining portion twenty minutes. The three portions 
are now united and distilled water is added to the original 
volume, to compensate for the loss by evaporation. The 
mixture is allowed sufficient time for the precipitate to 
settle (about an hour). Now add an equal volume of 0.5 
per cent, aqueous solution of yellowish eosin (filtered) to 
the methylene blue solution in a large evaporating dish. 

1 Wilson, T. M. "On the chemistry and staining properties of certain de- 
rivatives of the methylene blue group when combined with eosin. ' ' Jour. Exp. 
Med., 1907, IX, 645. 

2 The cheaper grades of methylene blue may be used with satisfactory 
result. 

3 The silver oxid may be prepared by dissolving 2.0 gm. of silver nitrate in 
15 c. c. of distilled water and adding to it 260 c. c. of calcium hydrate. Shake 
well, and set aside for the precipitate to settle. Decant the supernatant fluid, 
collect the precipitate on a filter, and wash with 20 to 25 c. c. of distilled 
water. Dry the precipitate at a temperature not exceeding 100° C, and place 
it in a brown bottle, tightly stoppered. 



THE BLOOD 285 

Mix the solutions well, and allow the mixture to stand one 
hour. Filter thrice, using a hard filter paper, such as the 
Schleicher and Schiill filter, No. 575, and finally wash the 
precipitate which has collected on the filter paper with 
physiological salt solution. (The precipitate which adheres 
to the evaporating dish is discarded.) Dry the precipitate 
in the thermostat, and transfer it to a dark bottle, tightly 
stoppered. The staining solution is then prepared by dis- 
solving 0.4 gm. of the powdered precipitate in 100 c. c. of 
absolute methyl alcohol (Kahlbaum's). The stain may be 
rubbed in a mortar with the alcohol to facilitate solution, 
or powder and alcohol are placed in a bottle, which is vigor- 
ously shaken a few minutes on several successive days. 
The staining solution should be preserved in a dark bottle 
with glass stopper. (Wilson advises the use of 0.3 gm. of 
the dry stain to 100 c. c. of denatured alcohol, but in our 
hands this has not given satisfaction.) It is best to make 
up small quantities of the stain at frequent intervals (3 to 
4 months). 

Method of Staining. — As the methyl alcohol in which 
the stain is dissolved is apt to run over the edge of the 
cover glass, it is advisable to use the usual wire staining 
forceps ; when staining on glass slides, the stain may be 
confined to the area of the smear by drawing lines on the 
glass with a blue wax pencil — the red wax is usually loos- 
ened by the alcohol. 

(a) Cover the unfixed blood film with 5 to 6 drops of 
the stain for 1 minute. As the stain is dissolved in abso- 
lute methyl alcohol, the blood is fixed by this procedure. 
Precipitation of the stain through evaporation of the al- 
cohol will be troublesome, if too little stain is used. 

(b) Add to the stain an equal number of drops of dis- 
tilled water, and allow it to remain on the film 2 to 4 min- 



286 FRESH AND STAINED PREPARATIONS OF BLOOD 

utes. A metallic scum forms on the surface. (The exact 
proportion of stain and water should be determined for 
each new lot of stain. At times twice the quantity of water 
is necessary; occasionally, however, fewer drops of water 
than of stain are required, especially with old mixtures, 
which have become slightly acid.) 

(c) Wash with distilled water, blot dry, and mount in 
neutral balsam. The specimen should be held level during 
the washing and the stream of water directed against the 
surface of the cover glass, so that the metallic scum and 
precipitate in the fluid will be floated off. Avoid dumping 
the stain from the cover glass, for the precipitate adheres 
to the corpuscles, and cannot be removed by washing in 
water. If there is precipitate in the specimen, it may be 
removed by immersing the preparation momentarily in ab- 
solute methyl alcohol or ethyl alcohol, but always at the 
risk of decolorizing the cells too much; it is particularly 
the basic stains, methylene blue and methylene azure, which 
are decolorized. 

A properly stained blood film should have a pinkish- 
gray or gray color when dry. If the color of the film is 
bright pink, the specimen is usually not satisfactory, or, 
rather, it is capable of being improved upon. The erythro- 
cytes are stained very pale pink (plate II) or mauve or 
grayish-pink. Polychromatophilia is denoted by varying 
admixtures of blue. In extreme grade a polychromato- 
philic red cell is dark blue, no trace of pink being discern- 
ible. Basophilic granules stain dark blue. Occasionally, 
particularly in the blood of pernicious anemia, fine gran- 
ules are seen which are stained violet or purple. Nuclei 
are stained purple. Nuclear particles stain like the nuclei, 
while the ring bodies are usually violet or reddish-purple. 
The differentiation of these abnormalities is more striking 



LEGEND FOR PLATE II. 

(All drawings made with camera lucida; X 1200. Wilson's stain, 
modified Romanowsky.) 

1. Normal red corpuscle. 

2. Pale or anemic corpuscle. 

3. Basophilic granules in erythrocyte. 

4. Nuclear particle (Howell's body) in erythrocyte. 

5. Erythrocyte containing nuclear particle and basophilic granules. 

6. Polychromatophilic red cell containing a Cabot's ring body. 

7. Normoblast. 

8. Slightly polychromatophilic erythrocyte containing a nuclear particle, 

Cabot's ring body, and violet colored basophilic granules. 

9. Normoblast showing an early stage of karyorrhexis. 

10. Megaloblast, markedly polychromatophilic. 

11. Poikilocyte, markedly polychromatophilic and exhibiting reddish 

basophilic granules. 

12. Blood platelets. 

13. 14. Small lymphocytes. 

15. Large lymphocyte, exhibiting azurophilic granules. 

16. Large mononuclear leukocyte with a few azurophilic granules in the 

C} r toplasm. 

17. " Transitional " leukocyte with fine azurophilic granules. 

18. Polynuclear neutrophilic leukocyte. 

19. Polynuclear eosinophilic leukocyte. 

20. Mast cell or polynuclear basophilic leukocyte. 

21. Neutrophilic myelocyte. 



PLATE II 




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i 



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12 



13 




14- 



15 





- 



O 



16 



17 



18 




* ' n 



19 



SO*' 




20 



£x* 



THE BLOOD 287 

and brilliant with the Romanowsky stains than with any 
of the other blood stains. Cabot's ring bodies are usually 
demonstrable only with Romanowsky stains, i. e., they are 
stained with methylene azure. 

Blood platelets are well brought out with Romanowsky 
stains alone. The granular chromatin masses are stained 
reddish-purple, the body of the platelet being unstained 
or exhibiting varying shades of blue. 

All leukocytic nuclei are beautifully stained, the color 
being a deep reddish-purple. The morphology of the nu- 
cleus is well demonstrated. The leukocytic granules, on 
the other hand, stain poorly and very uncertainly, so that 
differential counting may be difficult or well-nigh impos- 
sible when pathological cells are present. The neutrophilic 
granules are stained lilac, but often only a few of the gran- 
ules — sometimes none of them — take the stain. Eosinophi- 
lic granules are pink, but stain very uncertainly; at times 
there may be considerable doubt as to their nature. Bas- 
ophilic granules are colored purple. The granules of the 
myelocytes usually are purple, regardless of the variety of 
the cell, though eosinophilic myelocytes may show a shade 
of pink in the granules. The cytoplasm of lymphocytes is 
colorless or blue — at times a very dark blue. In about one- 
third of the lymphocytes of normal blood purplish granules, 
varying much in size and number, are evident in the cyto- 
plasm. These granules are demonstrable only with methy- 
lene azure, and are, therefore, designated azurophile gran- 
ules. The large mononuclears and transitionals are, like 
the lymphocytes, more beautifully demonstrated with Ro- 
manowsky stains than by any other means. The cytoplasm 
of the large mononuclears is colorless or blue and generally 
non-granular, though it is now and then seen to be filled 
with azurophilic granules, which are for the most part very 



288 FEESH AND STAINED PREPARATIONS OF BLOOD 

fine and dust-like. In the case of the transitional leuko- 
cytes the protoplasm is studded with very fine azurophilic 
granules practically without exception. When these gran- 
ules are observed in a large mononuclear cell, the resem- 
blance it bears to a myelocyte is close at first glance. It 
is seen, however, that, while similar in color, the myelocytic 
granules are rather coarser, and close inspection will usu- 
ally reveal the granules over the nucleus in the myelocyte, 
a point which serves to differentiate them from the large 
mononuclears. Usually, too, the relations between nucleus 
and protoplasm are different in the two types of cell. 
(With the triacid stain and with Jenner's stain this diffi- 
culty never arises, since the large mononuclears are non 
granular.) 

Besides precipitated stain in the specimen, which may 
be avoided as indicated above, the chief difficulty in the ap- 
plication of the Romanowsky stains arises in understating 
with the basic components of the mixture. When this oc- 
curs, the erythrocytes are bright pink or red. the nuclei of 
the leukocytes blue instead of purple, and the chromatin of 
the platelets blue or unstained, while the chromatin of ma- 
larial or other parasites is entirely unstained. Such a con- 
dition may be due to one or more causes: (1) Dilution of 
the stain with too much water interferes with the nuclear 
staining. The requisite proportions of stain and water 
must be learned by experiment. If the nuclei are poorly 
stained, try less water. (2) Even a trace of acid in the 
water used for diluting or washing will weaken or remove 
the basic stains more or less completely. Staining in a 
room in which there are acid fumes is at times sufficient to 
ruin the specimen. (3) Acidity of the staining mixture 
itself may explain the failure of the nuclear stain. A 
minute quantity of acid in the bottle (or on the cork) in 



THE BLOOD 289 

which the stain is placed, or the formation of formic acid 
from the methyl alcohol, are possible sources of difficulty. 
Staining mixtures which contain acid may be made perfect, 
according to Peebles and Harlow, 1 by the addition of a few 
drops of absolute methyl alcohol in which a small quantity 
of potassium hydrate (by alcohol) has been dissolved. In 
case too much alcohol has been added, as shown by over- 
staining with the blue, it may be cautiously titrated back 
with absolute methyl alcohol containing a trace of glacial 
acetic acid. (4) Prolonged washing may spoil the result. 
It can readily be demonstrated by experiment that washing 
in water tends to remove the basic (blue) elements of the 
stain, thus rendering the eosin more conspicuous. With a 
good staining mixture and the right proportion of stain 
and water, it is only necessary to wash long enough to re- 
move the excess of stain ; the specimen is then blotted dry 
to prevent further decolorization. 

Overstaining with the blue is seldom a source of diffi- 
culty, except in old smears. When it does occur, it may be 
corrected by one of the four procedures just described. 
With very old blood films, it may be impossible to prevent 
diffuse blue staining of the red corpuscles, though Brem's 
method (p. 175) or Giemsa's slow method may give good 
results. 

(b) Leishman's Stain. — This stain is most conveniently 
obtained in tablet form from Burroughs, Wellcome, & Co. 
or from Grtibler. Grtibler also supplies the powdered stain 
in bulk. A tablet is dissolved in a stated quantity of abso- 
lute methyl alcohol, and the mixture is then ready for use. 
It should be kept in a tightly stoppered bottle. 

The staining technique is practically identical with that 

Peebles, A. B., and Harlow, W. P. "Clinical observations of blood 
stains. ' ' Jour. A. M. A., 1909, LII, 768. 



290 FRESH AND STAINED PREPARATIONS OF BLOOD 

given for Wilson's stain. The nuclei have a little more of 
a reddish hue. but otherwise the picture is much the same. 
The advantages and limitations of the stain are those given 
above. 

(c) Giemsa's Stain. — The preparation of Giemsa's 
stain is difficult. Griibler & Co. supply a reliable solution. 
The staining mixture contains eosin. azure I. and azure 
II. 

Method of Staining. — (a) Fix the specimen in absolute 
methyl or ethyl alcohol. 

(b) Add one drop of the staining mixture to 1 c. c. of 
distilled water. | This must be freshly prepared. I Stain 
10 to 30 minutes with this dilution of the staining mixture. 

(c) Wash with distilled water, blot dry. and mount in 
balsam. 

The appearance of the stained film is the usual Roma- 
nowsky picture (see p. 286). The nuclei, however, are a 
little redder than usual, and the neutrophilic granules are 
even less uniformly stained than with most other Roma- 
nowsky stains. 

In case the chromatin staining is unsatisfactory, a 
very dilute solution of sodium carbonate may be substi- 
tuted for distilled water in preparing the dilution of the 
stain. 

Old Blood Films. — Blood films which have been kept 
for several months before staining them do not give good 
results with the usual Bomanowsky procedures. The diffi- 
culty lies chiefly in the staining of the erythrocytes, which 
take a diffuse, slate-blue color. To avoid this to a great 
extent, the films may be stained with Leishman's or "Wil- 
son's stain, using Brem's technique (p. 175). or with 
Giemsa's stain. With the latter the dilution of the stain 
should be one drop to five or more cubic centimeters of 



THE BLOOD 291 

water. The specimens are allowed to remain in this fluid 
24 to 48 hours. They are then washed in water and 
mounted as usual. With this procedure it is often possible 
to demonstrate the chromatin of malarial parasites in 
specimens a year or more old. 

(7) Jenner's Stain x (the eosinate of methylene blue).— 
If one stain alone were to be selected for the general rou- 
tine examination of the blood, Jenner's would probably 
be the choice of most workers. It is a much better stain 
for nuclei and for pathologic alterations in the red cor- 
puscles than Ehrlich's triacid, though inferior to the Eo- 
manowsky stains in these respects. On the other hand, it 
is much superior to the Romanowsky stains for the dem- 
onstration of the granules in leukocytes, though surpassed 
for this purpose by Ehrlich's triacid mixture. 

Prepakation of the Stain. — The tablets of Jenner's 
stain, which have been placed upon the market by Bur- 
roughs, Wellcome, & Co. and by Grubler, have done away 
with the necessity of making the stain, and have made it 
possible to have on hand fresh solutions of the staining 
mixture. The tablets are dissolved in a stated quantity 
of methyl alcohol; the solution is ready for use at once. 
Numerous modifications of Jenner's methods of preparing 
the stain have been described, without, however, simplifica- 
tion or improvement of the original procedures. Jenner's 
methods are as follows: 

(a) First Method. — Prepare a 1.2 per cent, to 1.25 per 
cent, solution of Grubler 's water-soluble, yellowish eosin in 
distilled water, and also a 1 per cent, aqueous solution of 
Grubler 's medicinal methylene blue. Mix equal parts of 
the two solutions in an evaporating dish and, after stirring 

1 Jenner, L. "A new preparation for rapidly fixing and staining blood. ' ' 
Lancet, 1899, I, 370. 



292 FRESH AND STAINED PREPARATIONS OF BLOOD 

thoroughly, allow the mixture to stand for 24 hours. Filter 
through a hard filter paper (Schleicher and Schiill's No. 
575), and dry the precipitate, which collects on the paper, 
either at room temperature or in the incubator at 37° C. 
The temperature may be as high as 55° C. without injuring 
the precipitate. The dried precipitate is removed from 
the filter paper, powdered in a mortar, shaken with dis- 
tilled water, and the precipitate again collected on a filter 
paper. The washings should have a dirty purplish color. 
Finally, the precipitate is again dried and stored in brown 
glass bottles. For use dissolve 0.5 gm. of the powdered 
precipitate in 100 c. c. of absolute methyl alcohol, filter, 
and preserve in a tightly stoppered bottle. The solution 
keeps well. 

(b) Second Method. — Instead of using aqueous solu- 
tions, Jenner found that the eosin and methylene blue may 
be dissolved directly in absolute methyl alcohol. The stain- 
ing mixture is prepared by adding 125 c. c. of a 0.5 per 
cent, solution of Griibler's yellowish eosin in absolute 
methyl alcohol to 100 c. c. of a 0.5 per cent, alcoholic solu- 
tion of Griibler's medicinal methylene blue. The mixture 
is ready for use immediately. 

Methods of Staining. — The following is the technique 
originally recommended by Jenner: 

(1) Cover the unfixed blood film with the stain 
1 to 3 minutes. To prevent evaporation and precipita- 
tion of the stain, the specimen is covered with a watch 
glass. 

(b) Wash quickly in distilled water, blot dry, and mount 
in balsam. 

A second method of staining, which often gives good 
differentiation of the granules of the leukocytes and poly- 
chromatic nuclear staining, is as follows: 



THE BLOOD 293 

(a) Cover the unfixed specimen with about 8 drops of 
stain for 2 to 3 minutes. 

(b) Add to the stain about 10 drops of distilled water, 
and allow the mixture to remain on the preparation 1 to 2 
minutes or longer. 

(c) Wash with distilled water (observing the precau- 
tions given on page 286), blot dry, and mount in balsam. 

With Jenner's stain the red blood corpuscles are terra 
cotta or pink. Polychromasia causes the cell to assume a 
bluish tint. Basophilic granules in the red cells are dark 
blue, nuclei and nuclear particles are of the same color, 
though usually somewhat more deeply stained. Cabot's 
ring bodies are unstained or pale blue. 

The blood platelets are poorly stained. They are mauve 
in color and indistinct morphologically. With the second 
method, however, they are well differentiated, as with 
Romanowsky stains. 

Leukocytic nuclei are stained dark blue (purple with 
the second method). The neutrophilic granules are red, 
often with a violet tint. Eosinophilic granules are also red, 
but they are distinguishable from the former by their 
greater size and brilliance of staining. Mast-cell granules 
are purple. The granular differentiation is less distinct in 
myelocytes than it is in polynuclear cells, as a rule, and is 
inferior to that obtained with Ehrlich's triacid stain. 

The protoplasm of malarial parasites is stained light 
blue; chromatin is usually unstained. 

1 In staining specimens on glass slides, where the area of the film is larger, 
more stain should be employed to prevent too rapid concentration of the stain 
through evaporation. The relative proportion of stain to water should be pre- 
served. 






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THE BLOOD 295 

Methyl Green-Pyronin Mixture of Pappenheim. 1 — Sat- 
urated aqueous solutions of methyl green and of pyronin 
are made. One part of the pyronin solution is added to 
three to four parts of methyl green. When sufficient pyro- 
nin has been added, the mixture begins to take on a bluish 
tint. It is often possible to obtain very good staining mix- 
tures from Grubler. 

Method of Staining. — (a) Fix the blood films with heat. 

(b) Stain 5 to 10 minutes. 

(c) Wash quickly in distilled water, blot dry, and mount 
in balsam. 

All nuclei are stained dark green or blue. Erythrocytes 
take a gray or slate color. Polychromatophilic cells are 
stained more or less intensely by the pyronin, and exhibit 
varying shades of red. Basophilic granules are stained a 
brilliant red, in marked contrast to the nuclei. Nuclear 
particles are stained dark green or blue, 2 like normoblastic 
nuclei, or red, like the basophilic granules. 

The protoplasm of lymphocytes is stained red. Leuko- 
cytic granules are not specifically stained. 

The Iodin Reaction of the Leukocytes.— This reaction, 
discovered by Ehrlich, is demonstrated as follows: (1) 
The air-dried blood films are placed in a mixture composed 

Iodin 1.0 gm. 

Potassium iodid 3.0 gm. 

Distilled water 100.0 c. c. 

Gum arabic, q. s. (to give a syrupy consis- 
tence). 

1 Pappenheim, A. ' ' Vergleichende Untersuchungen ueber die elementare 
Zusammensetzung des rothen Knochenmarkes einiger Saugethiere. ' ? Virchow 's 
Archiv, 1899, CLVII, 19. 

2 Morris, E. S. " Nuclear particles in the erythrocytes." Arch. Int. Med., 
1909, III, 93. (The observations reported here were made on old films. Sub- 
sequent study of fresh material shows that nuclear particles, while not infre- 
quently stained blue, are, nevertheless, often red.) 



296 FRESH AND STAINED PREPARATIONS OF BLOOD 

(2) The air-dried specimen, instead of being placed in 
the mixture given above, may be put in a small vessel, in 
which a few crystals of iodin have been placed. The re- 
action appears in a few minutes. The specimen is mounted 
and examined in syrup made of levulose. 

Permanent specimens cannot be made by either proce- 
dure. 

The erythrocytes are stained diffusely brown. 

A positive reaction consists in brown staining, of vary- 
ing degree, of the protoplasm of the polynuclear neutro- 
philes. Occasionally the lymphocytes and mast cells are 
stained, rarely the large mononuclears and eosinophiles, 
while myelocytes never give the reaction (Zollikofer). 

Differential Counting of the Leukocytes 

For a differential count of the leukocytes the first es- 
sential is a well-spread and stained blood film. Ehrlich's 
triacid or Jenner's stain should be employed in the major- 
ity of instances. When myelocytes are present in the blood 
Ehrlich's triacid should be used, though for the usual run 
of cases Jenner's stain gives satisfactory differentiation. 
The Eomanowsky stains are unsatisfactory for differential 
counting, for the granular leukocytes are often poorly dif- 
ferentiated. The non-granular cells are, however, beauti- 
fully stained. 

For special purposes the triacid or Jenner's stain may 
be inferior to others. In studying eosinopliilia, when it is 
desired simply to follow the percentage of eosinophiles 
from day to day, hematoxylin or methylene blue combined 
with eosin may be used, always at the risk, however, of 
missing certain abnormal cells, if present. In lymphoid 
leukemia, where the lymphocytes may constitute 90 per 



THE BLOOD 297 

cent, or more of the leukocytes, it is advantageous to use a 
better nuclear stain than the triacid, and one which will 
differentiate between nucleus and protoplasm more effec- 
tively than Jenner's. The Romanowsky stains are the 
best for this purpose, for the staining is sharp and clear. 
Hematoxylin also gives excellent results, though the pic- 
ture is less comprehensive, for all granules are unstained. 
The large mononuclears and transitionals are most satis- 
factorily demonstrated with the Romanowsky stains. The 
differentiation between nucleus and protoplasm is clear, 
the morphology of the nucleus is shown in great detail, 
and the azurophile granules are made evident. 

For the differential count a mechanical stage is almost 
a necessity. The stained specimen is placed on the stage 
and examined with the tV -in. oil immersion objective and 
an eye-piece (such as Leitz No. 3 or No. 4), which gives a 
high magnification. In making the count it is, of course, 
essential that the cells be counted only once. This is accom- 
plished through the use of the mechanical stage, the speci- 
men being moved up and down, with a lateral shifting of 
the field at the end of each "row." At least 500 cells 
should be counted. 

Normal Leukocytes.— The leukocytes of the blood may 
be classified as follows: 

(1) Lymphocytes (pi. I, 5; pi. II, 13, 14, 15) are cells 
having a single round or oval nucleus — rarely a kidney- 
shaped nucleus — and a rather scanty rim of protoplasm. 
The protoplasm is non-granular, though about 30 per cent, 
of the lymphocytes of normal blood possess azurophile 
granules, which are demonstrable after staining with meth- 
ylene azure, but not with other stains. These granules 
vary greatly in number and size. Often there are many 
granules, more frequently only a few, in a cell. The diam- 

21 



298 FRESH AND STAINED PREPARATIONS OF BLOOD 

eter of lymphocytes varies between 7 and 11 micra. They 
constitute 22 to 25 per cent, of the leukocytes normally. 

(2) Large mononuclear leukocytes (pi. I, 6; pi. II, 16) 
resemble the lymphocytes, but are larger and have rela- 
tively more protoplasm. The nucleus is somewhat poorer 
in chromatin and, therefore, stains less intensely. The 
protoplasm may contain azurophile granules, which are 
either very fine, like those of the transitionals, or rarely 
coarser, like the granules of the lymphocytes. The cells 
are actively ameboid and phagocytic — the macrophages of 
the blood. The diameter is 12 to 20 micra. 

(3) "Transitional" leukocytes (pi. I, 7; pi. II, 17) differ 
from the large mononuclears in the shape of the nucleus, 
which is horseshoe-shaped, lobulated, or deeply indented, 
and in the constant presence in the protoplasm of numerous 
fine, dust-like azurophile granules. With stains other than 
the Eomanowsky, both cells present non-granular cyto- 
plasm, though exceptionally a few fine, faintly stained 
granules are discernible after staining with Ehrlich's tri- 
acid stain. These granules, when evident, are usually 
stained a reddish or pinkish tint. 

The large mononuclears and transitionals together form 
about 3 to 5 per cent, of the leukocytes of normal blood. 
The diameter is the same as that of the large mononuclears. 

(4) Polynuclear neutrophilic leukocytes (pi. I, 8; pi. II, 
18) are cells with polymorphous nuclei, in whose cytoplasm 
are numerous fine, neutrophilic granules. These cells are 
about 9 to 12 micra in diameter and constitute 65 to 70 
per cent, of the white cells under normal conditions. The 
cells are actively ameboid and phagocytic in the fresh blood 
and are designated microphages, in distinction to the ma- 
crophages or large mononuclears. 

(5) Polynuclear eosinophilic cells (pi. I, 9; pi. II, 19) 



THE BLOOD 299 

are similar to the last group (4), except for the presence of 
coarse eosinophilic or acidophilic granules in the proto- 
plasm. They resemble the neutrophils in size and in the 
possession of ameboid activity. Normal blood contains 
about 2 to 4 per cent, of these cells. 

(6) Mast cells (pi. I, 10; pi. II, 20) are polynuclear 
basophilic leukocytes. The nucleus is usually simply in- 
dented or lobulated. The protoplasm contains basophilic 
granules, which are somewhat variable in size, the major- 
ity being about as coarse as the eosinophilic granules. 
The cells measure about 10 micra in diameter. Normally 
about 0.5 per cent, of mast cells are found in the blood. 

Pathological Leukocytes.— In addition to the foregoing 
cells, which go to make up the leukocytes of normal blood, 
there appear in disease immature cells, the precursors of 
the ripe leukocytes of normal blood. 

(7) Neutrophilic myelocytes (pi. I, 11; pi. II, 21) are 
the antecedents of the polynuclear neutrophilic cells. They 
differ from the latter in having a round or oval or slightly 
indented nucleus. The protoplasm contains neutrophilic 
granules, which are often finer than those of the polynu- 
clear cells. In the older myelocytes the granules are abun- 
dant, while very young cells may contain only a few. The 
nucleus is poorer in chromatin than that of the mature 
polynuclear cell. The cells are subject to great variation 
in size. The majority lie between 12 and 20 micra in diam- 
eter, though larger and smaller cells are encountered now 
and then. In the nucleus one to four nucleoli may be 
visible, particularly after staining with methyl green-pyro- 
nin or with Eomanowsky stains. The nucleus is usually 
eccentrically situated. 

(8) Eosinophilic myelocytes (pi. I, 12) resemble neu- 
trophilic myelocytes in every respect aside from the dif- 



300 FRESH AND STAINED PREPARATIONS OF BLOOD 

ference in the granules. Frequently the immature eosino- 
philic granules exhibit basophilic tendencies, in that they 
are stained with basic dyes. Thus, in a Ronianowsky prep- 
aration, some or all of the granules may take a dark blue 
or purplish tint. 

(9) "Mast" myelocytes (pi. I, 13) are generally small 
and present basophilic granules in the protoplasm. 

(10) Metamyelocyte is a term used to designate cells 
whose nuclei have passed beyond the kidney shape and 
already present more or less deep indentations. They are 
transition stages between the myelocyte and the polynu- 
clear neutrophilic cells. They are not to be confused with 
the so-called transitional cells (which are, in reality, mis- 
branded, as they represent transition forms to no type of 
cell, so far as is known, though it was originally supposed 
that they developed into the polynuclear neutrophiles, hence 
the name "transitional"). They are differentiated from 
the transitionals by the abundance of neutrophilic granules 
in their cytoplasm, when stained with Ehrlich's triacid 
or Jenner's stain. With Ronianowsky stains, on the other 
hand, the granules of the metamyelocyte and transitional 
may be identical in color, but those of the transitional are 
much finer. 

(11) Promyelocytes represent the earliest form of my- 
elocyte, the cell with very few granules in its cytoplasm. 
The term is superfluous. 

(12) Myeloblasts (non-granular marrow cells, undiffer- 
entiated cells of the marrow, lymphoid cells of the marrow, 
etc.) (pi. I, 14) are the parent cells of the myelocytes. 
They differ from the latter in the complete lack of cyto- 
plasmic granules. The nucleus is similar to that of the 
myelocyte. The protoplasm is basophilic. The cells vary 
from the size of a lymphocyte to cells 20 micra in diameter. 



THE BLOOD 301 

(13) Irritation forms (Tiirk) are cells with round or 
oval nucleus, like that of the myelocyte, and rather abun- 
dant protoplasm, which is markedly basophilic and gen- 
erally vacuolated. 

(14) Pathological lymphocytes. In disease lymphocytes 
may depart considerably from the normal. The size is 
subject to much greater variation; the protoplasm is often 
greatly reduced in amount and at times is not demon- 
strable. The nucleus may be convoluted or indented, the 
so-called Rieder cells. 

(15) Megakaryocytes, the giant cells of the bone mar- 
row, are very rare in the blood. The nucleus is greatly 
convoluted, and the cytoplasm, with Eomanowsky stains, 
exhibits fine, dust-like granules. The cells are very large 
in the bone marrow, but only the smaller examples pass the 
capillaries and appear in the circulating blood. 



The Normal and Pathological Red Blood Corpuscles 

Non-nucleated red cells are designated erythrocytes, 
the nucleated forms erythroblasts. 

Erythrocytes. — (l)Normocytes (pi. I, 1, 2; pi. II, 1) are 
normal red blood corpuscles. In the fresh specimen they 
present the familiar form of biconcave discs, the center 
being paler, owing to the thinner layer of hemoglobin at 
this point. The normal cells stain with acid dyes (ortho- 
chromatic). The average diameter is 7.5 micra. 

(2) Microcytes are abnormally small erythrocytes. 

(3) Macrocytes or megalocytes are abnormally large 
erythrocytes. They may be pale, swollen corpuscles or ab- 
normally rich in hemoglobin. The diameter may amount 
to 25 micra. Abnormal variation in size of the corpuscles 



302 FRESH AND STAINED PREPARATIONS OF BLOOD 

is designated anisocytosis. Ameboid movements may be 
observed in some of the cells. 1 

(4) Poikilocytes (pi. II, 11) are cells of irregular form. 
The shape may be practically anything. 

Erythroblasts. — Nucleated forms of the red blood cor- 
puscle may be subdivided as follows : 

(5) Normoblasts (pi. I, 4; pi. II, 7, 7, 9) are nucleated 
red corpuscles, having the diameter of the average normo- 
cyte. The nucleus is round, at times eccentric, and usually 
very dense (pyknotic) and rich in chromatin. Younger 
forms present nuclei with a visible chromatin network. 

(6) Microblasts are abnormally small, nucleated red 
cells. 

(7) Megaloblasts (pi. I, 3; pi. II, 10) are large erythro- 
blasts, possessing a large oval or round nucleus. The 
diameter of the nucleus exceeds that of a normal red cell 
(Emerson). In the youngest forms the nucleus exhibits a 
beautiful chromatin network, with markedly basophilic 
protoplasm. More mature megaloblasts are more nearly 
orthochromatic and the nucleus is more homogeneous, the 
network being less conspicuous or lacking. Mitoses may 
be observed. 2 Ameboid activity may be seen in fresh speci- 
mens. 3 

(8) Intermediates is a term used to designate all those 
nucleated red corpuscles which can be classified neither as 
normoblasts, megaloblasts, nor microblasts (Emerson). 

Abnormalities in the Staining of the Red Corpuscles.— 
(1) Polychromasia or polychromatopliilia (pi. II, 6, 8, 10, 

1 Morris, E. S., and Thayer, W. S. "Amoeboid movements in macrocytes 
and megaloblasts." Arch. Int. Med., 1911, VIII, 581. 

2 Dock, G. ' ' Mitosis in circulating blood. ' ' Trans. Assoc. Amer. Phys., 
1902. 

3 Thayer, W. S. "The amoeboid activity of megaloblasts." Arch. Int. 
Med., 1911, VII, 223. See also Morris, E, S. and Thayer, W. S. Loc. cit. 



THE BLOOD 303 

11) is a condition in which the red corpuscles stain with 
basic dyes. Normally the red cells possess an affinity for 
acid dyes, such as eosin. Polychromasia varies greatly in 
degree. With slight grades the color of the acid dye still 
predominates, though the tint of the basic dye is visible. 
When polychromasia is marked there is an intense staining 
of the cell with the basic dye alone. It occurs in both 
erythrocytes and erythroblasts. 

(2) Basophilic granulation or stippling (pi. II, 3, 5) is 
seen in non-nucleated red cells and in nucleated corpuscles, 
often with intact nuclei. As the name implies, the cell 
contains granules which are stained only with basic dyes. 
The granules are always quite numerous in the cell and 
vary considerably in size. As a rule, the smaller the gran- 
ules the more numerous they are. With the variation in 
size there is also irregularity of form. Generally the larger 
granules are observed in orthochromatic cells, the smaller 
in polychromatophilic cells, as Askanazy has pointed out. 

(3) Fragmentation of the Nucleus. — Karyorrhexis (pi. 
II, 9), the breaking up of the nucleus within the cell, leads 
to characteristic appearances. The fragmented nucleus 
may resemble a rosette, or the nucleus may resolve itself 
into a number of round or oval, often irregular, masses, 
which are united or separate from one another. 

(4) Nuclear Particles. — Nuclear particles (pi. II, 4, 5, 
8) are derived from the nucleus of the red cell through 
atrophy of the nucleus or by karyorrhexis ; it is possible 
that there is another mode of formation, since nuclear par- 
ticles, may be found in megaloblasts with active, intact nu- 
clei. They were first observed in the blood of the cat by 
Howell, 1 and are known as Howell's bodies or Howell's 

1 Howell, W. H. "The life-history of the formed elements of the blood, 
especially the red corpuscles." Jour. MorpJwl., 1890, IV, 57. 



304 FRESH AND STAINED PREPARATIONS OF BLOOD 

nuclear particles. They occur also in human blood. 1 They 
are small, round, sharply denned bodies, usually situated 
eccentrically in the cell, and generally occur singly, though 
as many as nine have been observed in a non-nucleated red 
corpuscle. They resemble miniature pyknotic nuclei mor- 
phologically. 

(5) Ring Bodies. — Eing bodies (pi. II, 6, 8) were first 
described by Cabot, 2 and are usually designated Cabot's 
ring bodies. The ring may remain round or may be twisted 
so as to form a figure eight, etc. The rings are best seen 
after staining with Eomanowsky stains, which usually color 
them red or reddish-violet, rarely blue. It is believed by 
Cabot, and by all who have since studied these bodies, that 
they represent the nuclear membrane. 

(6) "Red Basophilic Granulation with Romanowsky 
Stains" (pi. II, 8, 11). — Naegeli 3 in particular has called 
attention to the existence of a granulation seen in speci- 
mens stained with Giemsa's stain; it is demonstrable 
with all Romanowsky stains. The granules differ from 
the usual basophilic granules, in that they are stained 
red or violet instead of blue. The granules were ob- 
served by Cabot in cells which also contained ring bodies. 
Naegeli believes that the granules originate from the 
nucleus, probably from the nuclear membrane, since 
ring bodies may be made of a series of dots similarly 
stained. 

(7) Schiiffner's Granules. — Schiiffner's granules are 

1 Morris, E. S. (a) "Note on the occurrence of Howell's nuclear par- 
ticles in experimental anemia of the rabbit and in human blood. ' ' Johns 
Hopkins Eosp. Bull, 1907, XVIII, 198. (b) "Nuclear particles in the erythro- 
cytes. ' ' Arch. Int. Med., 1909, III, 93. 

2 Cabot, E. C. "Eing bodies (nuclear remnants'?) in anemic blood." 
Jour. Med. Eesearch, 1903, IX, 15. 

3 Naegeli, O. " Blutkrankheiten und Blutdiagnostik. " Leipzig, 1912, 
2nd Ed., p. 153. 



THE BLOOD 305 

found only in certain cases of malaria. They are seen in 
the infected corpuscles (see below). 

Demonstration of Protozoa in the Blood 

The blood protozoa which are of pathological impor- 
tance are few in number. At the present time the Plas- 
modia of malaria alone demand general consideration in 
this country. For protozoa in general, however, such as 
trypanosomes, Leishman-Donovan bodies, etc., the method 
of demonstration in the stained specimen, which is uni- 
versally employed, is one of the numerous modifications 
of the Romanowsky stain (q. v.). 

Malarial Parasites. — The plasmodia of malaria are 
characteristically stained by the Romanowsky method. 
The protoplasm of the parasite is stained light blue, con- 
trasting well with the pink color of the red corpuscle. The 
nuclear chromatin of the parasite is colored a brilliant red 
or purplish-red, while the pigment retains its original color, 
being unstained. 

In certain of the infections with Plasmodium vivax and 
Plasmodium falciparum, peculiar granulations appear in 
the infected red corpuscles (SchufTner's granules). They 
have been described by SchufTner and others. With Ro- 
manowsky stains, the granules exhibit a dark, reddish tint, 
often quite like that of the chromatin of the parasite. 
S chuff ner's granules are not to be confused with the ordi- 
nary basophilic granules of the red cells or with the red 
granulations seen in certain corpuscles when stained with 
Romanowsky stains. By means of vital staining Boggs * 
has adduced further proof of the non-identity of Schuff- 

1 Boggs, T. E. "Vital staining of ' stipple cells' in malarial blood." 
Jour. A. M. A., 1911, LVII, 150. 



306 FRESH AND STAINED PREPARATIONS OF BLQOD 

ner's with other granules. The granules may be missed 
in cells containing the youngest hyalin parasites, and usu- 
ally seem to increase in number with the age of the para- 
site. SchufTner's granules have not been observed in cells 
infected with the parasite of quartan fever. 

Blood platelets have been mistaken for hyalin forms of 
the plasmodia by inexperienced observers. This is apt to 
occur only when the platelet rests upon the red corpuscle. 
Differentiation is simple. The chromatin of the platelet 
is usually colored purple with less of the reddish tint than 
the chromatin of the parasite shows, but this difference 
may be lacking, for often the chromatin of the parasite is 
stained exactly the shade of that of the platelet. The im- 
portant differential point is found in the arrangement of 
the chromatin. In the platelet the chromatin is scattered 
in minute granules, while the chromatin of the hyalin para- 
site is in a compact mass, or, in the case of Plasmodium 
falciparum, in two or three masses, but still dense and 
compact. The body of the platelet is often unstained, but 
may take a pale blue color, very much like that of the proto- 
plasm of the parasite. Giant blood platelets have been mis- 
taken for extracellular forms of the malarial parasite. The 
constant presence of pigment granules in the parasite 
should be sufficient to differentiate, even though the dis- 
tribution of the chromatin in this instance be somewhat 
similar in the two — which is generally not the case. 

Staixixg Method of Ross. 1 — Ross has devised a method 
for detecting the parasites which is useful when their num- 
ber in the blood is small. He prepares a thick smear of 
the blood and, before staining the film, extracts the greater 
part of the hemoglobin from the cells. A drop of blood of 

1 Eoss, E. ''An improved method for the microscopical diagnosis of in- 
termittent fever." Lancet, 1903, I, 86. 



THE BLOOD 307 

about 20 c. mm. is placed on a cover glass (% in. sqnare) 
and spread in the usual manner, or with a needle or lancet. 
It is dried in the air. The preparation contains about 
twenty times the amount of blood usually found in a smear. 
After becoming dry it is covered with 1 per cent, eosin so- 
lution (Romanowsky's). The stain is placed on the speci- 
men with a glass rod and is allowed to act for as much as 
fifteen minutes. It is then washed. The washing must be 
done with a very gentle stream, since the unfixed blood is 
easily loosened. Then stain with Romanowsky's methylene 
blue x a few seconds and again wash carefully. The speci- 
men is dried in the air and mounted in balsam. The hemo- 
globin is extracted from the red corpuscles, leaving only 
their stromata, together with leukocytes, platelets, and 
Plasmodia. The staining of the parasite is distinctive — 
protoplasm light blue and nuclear chromatin red. Hyalins 
are readily detected. 

For finding larger, pigmented parasites Ross prepares 
the film as described above; after it has dried, it is cov- 
ered with distilled water to extract the hemoglobin. The 
unstained specimen is then examined. Crescents and other 
pigmented forms stand out prominently. 

Ruge's Modification 2 of the Method of Ross. — A dis- 
advantage in the method of Ross is the difficulty of wash- 
ing the unfixed specimen without losing the preparation. 
To overcome this, Ruge fixes the thick films in 2 per cent, 
formalin containing y 2 to 1 per cent, acetic acid. The 
hemoglobin is extracted from the erythrocytes, which are 
fixed to the cover glass at the same time. The specimen 

1 Nocht 's solution of methylene blue for the Romanowsky stain consists of 
methylene blue (rectif. puriss. Hoechst) 1.0 gm., sodium carbonate 0.5 gm., 
dissolved in 100 c. c. of distilled water. 

2 Ruge, R. ' ' Zur Erleichterung der mikroskopischen Malariadiagnose. ' ; 
Deutsche med. Wchnschr., 1903, XXIX, 205. 



308 EXAMINATION OF BLOOD FOR ANIMAL PARASITES 

is then stained with Romanowsky's stain. The formalin 
fixation interferes somewhat with the staining of the pro- 
toplasm of the plasmodia, so that it may be necessary to 
restain the film with methylene blue. A certain amount 
of precipitate remains in the specimen, but the parasites 
are well stained. 

Examination of the fkesh blood, whenever pos- 
sible, is the most satisfactory method of diagnosis of 
malaria, as the variety of the parasite is more easily recog- 
nized, as a rule, than in stained smears. The accompany- 
ing table gives the main differences between the three spe- 
cies of plasmodia in fresh blood ; it may be used also, with 
certain obvious exceptions, in connection with stained 
smears. 

Degenerations in the red cells may be mistaken for hy- 
alin parasites. In form the degenerations may bear a strik- 
ing resemblance to ring forms or irregularly shaped para- 
sites. Ameboid activity is lacking, and it may be noted 
that the degenerations become more numerous in the speci- 
men as time advances. The larger round or oval degenera- 
tions are less confusing; their size appears to change on 
raising and lowering the focus. 



Examination of the Blood for Aniw,al Parasites 

(1) Filaria bancrofti (Fig. 10), when present in the 
blood, is usually demonstrable by the ordinary method of 
examination of the fresh blood. The size of the drop should 
be a little larger than usual, in order to secure a moderately 
thick preparation. The blood should be taken during the 
sleeping hours — usually at night, when the embryos are 



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310 EXAMINATION OF BLOOD FOR ANIMAL PARASITES 

present in the blood vessels of the skin. The wriggling 
motion of the embryos arrests attention at once. Fair 
permanent specimens may be secured by making a rela- 
tively thick smear of the blood, which is then stained with 
hematoxylin and eosin, or one of the Romanowsky stains. 
Staubli 's method is useful when the parasites are scarce. 
The embryos are 0.125 to 0.300 mm. long and 0.007 to 0.011 
mm. thick (Blanchard). 

(2) Trichinella Spiralis.— The embryos of Trichinella 
spiralis are at times found in the blood. The method of 
detecting their presence is that which Staubli 1 evolved 
in the study of experimental trichinosis, and which has 
been successfully applied to the diagnosis of human infec- 
tions by Herrick and Janeway 2 and others. In human 
cases it is apparently desirable to employ larger quanti- 
ties of blood than Staubli found necessary. 

Method of Staubli. — By means of an aspirating syringe, 
1 to 10 c. c. of blood are withdrawn from an arm vein with 
the usual aseptic technique. The blood is immediately 
laked by adding it to about 15 volumes of 3 per cent, acetic 
acid, shaking thoroughly to prevent clotting. The acid is 
strong enough to insure complete taking of the erythro- 
cytes, and at the same time is not injurious to the embryos. 
The mixture is centrifugalized, the sediment removed with 
a pipette, and examined fresh. It consists mainly of leuko- 
cytes. The specimen should be examined with a mechanical 
stage, so that all parts of it may be inspected if necessary. 
Moderately high magnification should be employed. At the 
time of their entrance into the circulation the embryos are 

1 Staubli. C. "Beitrag zum Xaclrweis von Parasiten im Blut. ' ' MiincJien. 
med. Wchnschr., 1908, LV, 2601. 

2 Herrick, W. W., and Janeway, T. C. "Demonstration of the Trichinella 
spiralis in the circulating blood in man." Arch. Int. Med., 1909. Ill, 263. 



THE BLOOD 311 

about 0.120 to 0.160 mm. long and 0.007 to 0.008 mm. in 
thickness (Blanchard). 

Staubli has obtained good permanent specimens by 
staining the sediment with Giemsa's or Jenner's stain. The 
acetic acid should be completely removed by washing before 
attempting to use either of these stains. 



CHAPTEE VI 
PUNCTURE FLUIDS 

The methods described below are applicable to most 
puncture fluids, e. g\, pleural, peritoneal, pericardial, hy- 
drocele, etc. The examination of the cerebrospinal fluid, 
however, requires certain special procedures, which are 
considered separately. 

Specific Gravity.— The specific gravity is usually deter- 
mined only approximately with the urinometer. In filling 
the cylinder, special care is required to avoid bubbles on 
the surface because of the albuminous nature of the fluid. 
The determination should be made at once, before clotting 
of the fluid will have occurred. Because of the high tem- 
perature of the fluid immediately after withdrawal (ap- 
proximately that of the body), a correction of the urinom- 
eter reading should be made. Most instruments are stand- 
ardized for a temperature of 15° C. For each three (3) 
degrees C. above this temperature the specific gravity is 
depressed 0.001. The result is that the reading obtained 
on a perfectly fresh fluid is too low. (It must be remem- 
bered, however, that the values obtained with the clinical 
urinometer are, at best, only approximately correct.) 

Albumin Content.— Albumin content refers to the en- 
tire coagulable protein. For clinical use, the best method 
of determining the quantity of the coagulable protein is 
Tsuchiya's modification of the Esbach method (p. 35). 
It is necessary to dilute the fluid to such an extent that 
the reading in the Esbach tube will be 4 or less; in other 

312 



PUNCTURE FLUIDS 313 

words, the dilution must reduce the albumin below 0.4 per 
cent. With higher percentages of protein the method is 
subject to considerable error. The diluted fluid is weakly 
acidified with acetic acid, and the determination is carried 
out in the same manner as with urine. The reading at 
the end of 24 hours is multiplied by the dilution, and the 
result is the number of grams of coagulable protein per 
1,000 c. c. of fluid. 

Tsuchiya's method is less accurate with puncture fluids 
than with urine. 1 

Accurate determinations of the coagulable protein may 
be made in connection with the estimation of the incoagu- 
lable nitrogen (see below). Five c. c. of the puncture fluid 
(before clotting has occurred) are measured by means of 
a pipette into each of two Kjeldahl flasks. To the fluid in 
each flask add about 15 c. c. of concentrated sulphuric acid, 
about 0.2 gm. of copper sulphate crystals, and about 10 gm. 
of potassium sulphate. The usual Kjeldahl determination 
of total nitrogen is now made (p. 20). The distillate is 
collected in 50 c. c. of — sulphuric acid. The total ni- 
trogen in grams per cent, is calculated. From this the 
incoagulable nitrogen in grams per cent, is subtracted. The 
difference between the two, multiplied by the factor 6.25, 
gives the coagulable protein in grams per cent. 

The Incoagulable Nitrogen.— The accurate determina- 
tion of the incoagulable nitrogen in puncture fluids is too 
time-consuming at present to be applied generally by clin- 
icians. The method, an adaptation from Hohlweg and 
Meyer, is as follows : 2 To 10 c. c. of the puncture fluid in 
a 300-c. c. Erlenmeyer flask add a reagent composed of 

*Mattice, A. F. Personal communication. 

2 Morris, E. S. ' ' The incoagulable nitrogen of puncture fluids, with special 
reference to cancer. A preliminary note." Arch. Int. Med., 1911, VIII, 457. 
22 



314 THE INCOAGULABLE NITROGEN 

equal parts of 1 per cent, acetic acid and 5 per cent, solu- 
tion of monocalcium phosphate, until the reaction is acid 
to litmus, but still neutral to Congo red. The limit is 
rather wide, varying from 2 to 6 or more c. c. with differ- 
ent fluids. The amount must be determined separately for 
each fluid. Distilled water is now added to bring the vol- 
ume to 80 c. c, and then 80 c. c. of saturated aqueous solu- 
tion of sodium chlorid are poured into the flask. The mix- 
ture is now boiled to precipitate the coagulable proteins, 
and is then filtered through a folded filter (Schleicher and 
Schiill's No. 589, blue ribbon) directly into a Kjeldahl flask. 
The Erlenmeyer flask and filter are washed three times 
with distilled water. A Kjeldahl nitrogen determination 
is made on the filtrate and washings. Owing to the quan- 
tity of sodium chlorid contained in the filtrate, a consid- 
erable excess of sulphuric acid must be added to convert 
the sodium chlorid into sodium sulphate and still leave 
sufficient sulphuric acid for the oxidation. For this pur- 
pose about 30 c. c. of concentrated acid are enough. Be- 
cause of the preformed sodium sulphate, it is unnecessary 
to add potassium sulphate. After adding about 0.2 gm. 
of copper sulphate crystals, the oxidation and distillation 
are carried out as described under Kjeldahl nitrogen de- 
termination (p. 20). For collecting the distillate 15 c. c. 
of tenth normal sulphuric acid are used. All determina- 
tions are made in duplicate or triplicate, after a prelim- 
inary test of the filtrate with heat and dilute acetic acid 
has shown that the proteins are completely removed. 

The nitrogen is calculated in grams per cent. 

In the vast majority of fluids it will be found that the 
incoagulable nitrogen is below 0.0699 gm. per cent. — usu- 
ally considerably below this figure. Values between 0.07 
and 0.0899 gm. per cent, are of doubtful significance, but, 



PUNCTURE FLUIDS 315 

when the incoagulable nitrogen exceeds 0.09 gm. per cent., 
the probability is strong that the fluid is either cancerous 
or sarcomatous in origin. It should be kept in mind, how- 
ever, that lower values by no means exclude malignant 
disease. 

A Protein Precipitable in the Cold by Dilute Acetic 
Acid.— Euneb erg 1 was the first to apply to the diagnosis 
of puncture fluids Paijkull's observation on the presence 
of a protein precipitable by dilute acetic acid in the cold. 
He found that inflammatory exudates and those resulting 
from malignant neoplasms of the serous membranes usu- 
ally contain this body in abundance, whereas transudates 
do not, or at most possess it only in traces. His observa- 
tions have been confirmed by Umber, 2 Stahelin, 3 and others. 
Umber has designated the body in question "serosamucin. ,, 

Method. — About 10 c. c. of the fluid in a test tube are 
treated with a few drops of dilute acetic acid (3 per cent.) 
until the reaction becomes acid to litmus. A positive re- 
action is denoted by the appearance of a rather marked 
cloudiness. A slight cloud is of no significance, and is 
seen in transudates frequently. Previous clotting of the 
fluid apparently does not interfere with the test. A great 
excess of acetic acid may redissolve the precipitate, and is, 
therefore, to be avoided. 

Cytology. — As is the case with the blood leukocytes, 
cells are occasionally seen in exudates and transudates 
which cannot be definitely classified, but, for the most part, 

1 Kuneberg, J. W. ' ' Von der diagnostischen Bedeutung des Eiweissge- 
haltes in pathologischen Trans- und Exsudaten." Berlin. Tclin. Wchnschr., 
1897, XXXIV, 710. 

2 Umber, F. (a) ' * Ueber autolytische Vorgange in Exsudaten. ' ' Munchen. 
med. Wchnschr., 1902, XLTX, 1169. (b) "Zum Studium der Eiweisskorper in 
Exsudaten." Ztschr. f. Tclin. Med., 1903, XLVIII, 364. 

3 Stahelin, E. ' ' Ueber den durch Essigsaure f allbaren Eiweisskorper der 
Exsudate und des Urins. ' ' Munchen. med. Wchnschr., 1902, XLIX, 1413. 



316 CYTOLOGY 

the cells of puncture fluids may be placed in the following 
groups : 

(1) Lymphocytes. — Often cells similar to, and, doubt- 
less, identical with, the small lymphocytes of the blood are 
numerous in puncture fluids. They are characterized by 
their small size, relatively large nucleus, surrounded by a 
narrow rim of protoplasm. Frequently the protoplasm is 
so scanty that the nucleus appears to be, and sometimes is, 
naked. All transitions in size from the lymphocyte to the 
endothelial cell may be observed. 

(2) Endothelial Cells. — These are usually very large 
cells, with abundant protoplasm and one or more round or 
oval nuclei, rather poor in chromatin. Frequently several 
cells are seen en masse. The size of the cells, as well as 
their shape, is variable ; smaller forms are not infrequent. 
The cytoplasm may exhibit degenerative changes. 

(3) Poly nuclear neutrophile cells are similar to those 
of the blood, which is their source. They are often well 
preserved, but degenerations are common both in proto- 
plasm and nucleus. The polymorphous nucleus may come 
to resemble a single round nucleus. 

(4) Eosinophile cells are much less commonly met with 
than any of the foregoing. They may, at times, form a 
striking feature of the cell picture. 

(5) Mast cells are rare in puncture fluids. 

(6) Tumor cells, as such, are not recognizable. The 
presence of many mitotic nuclei, however, as Dock 1 was 
among the first to point out, is highly suggestive. Frag- 

1 Dock, G. "Cancer of the stomach in early life and the value of cells in 
effusions in the diagnosis of cancer of the serous membrane. ' ' Amer. Jour. 
Med. Set, 1897, CXIII, 655. Also Warren, L. F. "The diagnostic value of 
mitotic figures in the cells of serous exudates." Arch. Int. Med., 1911, VIII, 
648. 



PUNCTURE FLUIDS 317 

ments of tissue, when obtained, should be hardened and 
studied in section. 

(7) Red blood corpuscles, often well preserved, though 
frequently crenated or otherwise degenerated, are often 
found in puncture fluids, particularly when of tuberculous 
or malignant origin. 

Method of Obtaining Cells. — The fluid should be cen- 
trifugalized at high speed before clotting has occurred. 
The cells are then removed from the bottom of the cen- 
trifuge tube with a pipette, spread on glass slides, dried, 
fixed, and stained. One of the Romanowsky stains, Jen- 
ner's stain, or hematoxylin and eosin may be employed. 

In case the fluid cannot be centrifugalized immediately 
after its withdrawal from the body, about 10 c. c. of it 
should be discharged into an equal volume of 1 per cent, 
sodium fluorid solution to prevent clotting. For the sodium 
fluorid there may be substituted a 1.5 per cent, solution of 
sodium citrate in 0.85 per cent, sodium chlorid. Exam- 
ination of the cells should be made within a day. 

Tubercle bacilli may be sought in puncture fluids, but 
generally without success. Inoculation of the fluid, ob- 
tained under aseptic precautions, into the peritoneal cav- 
ity of a guinea-pig or rabbit is the best method of deter- 
mining the presence or absence of tubercle bacilli. About 
10 c. c. of fluid are injected. It is important that the fluid 
be injected before clotting has begun. Young animals are 
somewhat more susceptible to infection. After an interval 
of two to six weeks, the animal is sacrificed, and autopsy 
is performed, to determine the presence or absence of tu- 
berculous lesions. 



318 CEREBROSPINAL FLUID 



CEREBROSPINAL FLUID 



Lumbar puncture often yields only a few cubic centi- 
meters of cerebrospinal fluid, though at times 50 c. c. or 
more may be safely withdrawn. The determination of spe- 
cific gravity, which is normally between about 1.006 and 
1.010, is of little importance clinically. The number and 
kind of cells, the globulin content, and the bacteriological 
findings are the chief points of interest. 

Cells of the Cerebrospinal Fluid.— The cerebrospinal 
fluid normally contains very few cells. Indeed, some ob- 
servers have reported no cells in the normal fluid, though 
such a finding is probably exceptional. From 1 to 7 cells 
per cubic millimeter has been given as the normal limit. 1 
More than 10 cells per c. mm.- is pathological. 2 The cells 
are chiefly lymphocytes. In disease pus cells may be nu- 
merous; endothelial cells, eosinophiles, mast cells, and 
erythrocytes may be seen. 

Method of Counting the Cells. 1 — A saturated aqueous 
solution of methyl violet (5B) is drawn up in the tube of 
a 1 :100 blood pipette, until it has filled four of the decimal 
divisions on the capillary tube, and then the pipette is 
filled with the fresh spinal fluid, which should have been 
well shaken just before making the dilution to insure a 
uniform suspension of the cells. The leukocytes are stained 
violet. The pipette is shaken three minutes, and the count 
is then made with the blood-counting chamber, observing 

*Eous, F. P. "-Clinical studies of the cerebrospinal fluid, with especial 
reference to pressure, protein-content, and the number and character of the 
cells." Amer. Jour. Med. Sci., 1907, CXXXIII, 567. 

2 McCampbell, E. F., and Eowland, G. A. "Studies on the clinical diag- 
nosis of general paralysis of the insane." Jour. Med. 'Research, 1910, XXII, 
169. 



PUNCTURE FLUIDS 319 

the usual technique for counting blood. Emerson 1 recom- 
mends the use of the leukocyte pipette. The capillary tube 
is filled to the mark 0.5 with Unna's polychrome methylene 
blue, then to the mark 11 (or 21) with the fresh cerebro- 
spinal fluid. With this procedure more cells are counted. 

At times, in performing lumbar puncture, blood becomes 
mixed with the cerebrospinal fluid, the result being that 
the blood leukocytes raise the count of the cells of the 
cerebrospinal fluid. In such case the red cells in the fluid 
are counted, then both red and white cell counts of the 
blood are made. From the latter one obtains the relative 
number of red and white cells which have been introduced 
into the cerebrospinal fluid, and the count of the latter may 
be corrected. In making such correction it is essential that 
there shall have been no laking of the erythrocytes in the 
cerebrospinal fluid. 

Differential Count of the Cells. — The fresh fluid is cen- 
trifugalized, and the sediment is transferred with a pipette 
to clean glass slides, on which it is spread. The dried films 
are fixed and stained r usually with a Romanowsky stain. A 
differential count of the cells is then made 2 (see p. 296). 

Bacteriology of the Cerebrospinal Fluid.— Culture me- 
dia, preferably blood agar, should be inoculated with the 
fluid. (For further details see the works on bacteriology.) 
Smears are also made and examined. The Micrococcus 
intracellular is meningitidis, Diplococcus pneumonice, and 
Bacillus influenza are among the most important organisms 
encountered. 

Bacillus tuberculosis is demonstrable in the great ma- 
jority of instances of tuberculous meningitis. Several cu- 

1 Emerson, C. P. "Clinical Diagnosis." 3rd Edition, 1911, p. 700. 

2 Szeesi, S. "Neue Beitrage zur Cytologie des Liquor cerebrospinalis : 
Ueber Art und Herkunft der Zellen. " Ztschr. f. d. ges. Neurol, u. Psychiat., 
1911, VI, 537. 



320 CEREBROSPINAL FLUID 

bic centimeters of the fluid are placed in a sterile test tube. 
which is put in the refrigerator for 12 to 24 hours. A very 
delicate, filmy clot forms. This enmeshes the majority of 
the tubercle bacilli. The clot is then transferred to a clean 
slide, spread out into a thin layer with needles, air-dried, 
and fixed with heat. The specimen is then stained by the 
Ziehl-Neelsen method for tubercle bacilli. "With this method 
Heinenway 1 has demonstrated the organisms in a large 
series of cases. 

A second method of searching for the tubercle bacillus. 
which gives less constant results, is to centrifugalize the 
fluid at high speed until the sediment is thrown down 
(about 2.000 revolutions for 1 2 hour) ; it is then transferred 
to slides and examined in the usual manner for tubercle 
bacilli. 

In case of negative findings, several cubic centimeters 
of the fluid may be injected into the peritoneal cavity of 
a guinea-pig (see p. 317). 

Globulin Content.— Globulin is the most important 
protein of the cerebrospinal fluid. It is often present in 
increased quantity in disease, and may be detected by the 
following tests. Fluids with an admixture of blood can- 
not be used, since the serum globulin vitiates the tests. 

(1) Method of Xoguchi. 2 — To 0.1 c. c. of spinal fluid add 
0.5 c. c. of a 10 per cent, solution of pure butyric acid in 
0.9 per cent, sodium chlorid and boil briefly over the flame. 
Then add quickly 0.1 c. c. of y sodium hydroxid and again 

1 Hemenway. J. ' ' The constant presence of tubercle bacilli in the cerebro- 
spinal fluid of tuberculous meningitis." Amer. Jour. Vis. Child., 1911, I, 37. 

- Xoguchi, H. ' ' The relation of protein, lipoids, and salts to the Wasser- 
mann reaction." Jour. Exp. Med., 1909. XI, 84. Xoguchi, H., and !Moore, 
J. W. "The butyric acid test for syphilis in the diagnosis of metasyphilitic 
and other nervous disorders." Ibid., 1909. XI. 601. See also Strouse. S. 
"The diagnostic value of the butyric acid test (Xoguchi) in the cerebrospinal 
fluid." Jour. A, If. A.. 1911, LVT. 1171. 



PUNCTURE FLUIDS 321 

boil for a few seconds. With an increase of globulin a 
coarse granular or flocculent precipitate appears, usually 
within 10 to 20 minutes. If the precipitate does not appear 
within this time, the test tube is set aside and observed at 
the end of three hours. Normal fluids or those in which 
globulin is not increased give rise to a slight and uniform 
opalescence only, and no coarse precipitate forms, even af- 
ter several hours. In case of an ambiguous reaction, No- 
guchi advises a repetition of the test with 0.2 c. c. of spinal 
fluid. 

(2) Method of Eoss and Jones. 1 — Two c. c. of a satu- 
rated aqueous solution of ammonium sulphate are placed 
in a test tube, and 1 c. c. of the spinal fluid is gently run 
onto the surface of it, while the tube is inclined, so as to 
form a layer above the ammonium sulphate. The forma- 
tion of a thin, grayish-white ring at the line of contact of 
the two fluids constitutes a positive reaction. The pre- 
cipitate should form within three minutes. Within one- 
half hour it may be observed that the surface of the ring 
shows a delicate mesh appearance resembling a fine cob- 
web. The ring should be looked for with indirect illumina- 
tion, the tube being held against a dark background with 
the eye at a right angle to the source of light. It is essen- 
tial that the ammonium sulphate be quite neutral, not acid, 
and that the solution be saturated. 

The Wassermann Reaction in Cerebrospinal Fluid.— In 
syphilitic and metasyphilitic disease of the central nervous 
system the fluid obtained at lumbar puncture may yield a 
positive Wassermann reaction. 

1 Eoss, G. W., and Jones, E. "On the use of certain new chemical tests 
in the diagnosis of general paralysis and tabes." British Med. Jour., 1909, I, 
1111. 



INDEX 



Acetic acid in gastric contents, 143 
Aceto-acetic acid, 65. See also 

Diacetic acid 
Acetone in urine, 62 
Gunning's test for, 62 
Lange's test for, 64 
Legal's test for, 64 
Lieben's test for, 63 
Acholic stools, 154 
Acid alcohol, 213 
Acidity of gastric contents, 136 

of urine, 5 
Actinomyces bovis, 221 
Albumin in puncture fluids, 312 
in urine, 28-36 
in chyluria, 86 
in pyuria, 104 
qualitative tests for: 
heat and acetic acid, 28 
heat and nitric acid, 31 
Heller's test, 32 
potassium ferrocyanid and 
acetic acid, 34 
quantitative determination of, 

35 
removal of, from urine, 36 
Albuminuria, false, 104 
Albumose in urine, 37 
Aldehyde test of Ehrlich, 70 
Alimentary levulosuria, 56 
Alizarin red, 13 
Alkaptonuria, 61 

Almen-Nylander's test for sugar, 
40 



Alveolar cells in sputum, 209 
Amebse in feces, 172-176 

in sputum, 224 
Ammonia in urine, 12 

Folin's method for determination 

of, 13 
formalin titration method for de- 
termination of, 16-19 
Schlosing's method for deter- 
mination of, 19 
Shaffer's method for determina- 
tion of, 15 
Ammonio-magnesium phosphate in 
feces, 170 
in gastric contents, 151 
in sputum, 212 
in urine, 98 
Ammonium biurate in urine, 98 
Amylase (diastase) in feces, 163 

in urine, 87 
Anilin-water gentian violet, 218 
Auisocytosis, 302 
Ankylostoma duodenale, 184 
Antiformin for detecting tubercle 
bacilli, 214-217 
Boardman's method, 217 
composition of, 216 
Loeffler's method, 215 
Paterson's method, 216 
Areometer, Lohnstein's, 51 
Arnold's test for diacetic acid, 66 
Ascaris lumbricoides, 190 
Ascaris mystax (Toxocara canis), 
191 



323 



324 



INDEX 



Ascaris vermicularis (Oxyuris ver- 
micularis), 188 

Autenrieth - Konigsberger colori- 
meter, 122 

Azurophilic granules in large 



mononuclear 

298 
in lymphocytes, 297 
in transitionals, 298 



B 



leukocytes, 



Bacillus diphtherias, 219 
Bacillus influenzae, 219 
Bacillus tuberculosis, antiformin 
for detection of, 214 
in cerebrospinal fluid, 319 
in feces, 168 
in puncture fluids, 317 
in sputum, 212 
in urine, 115 

Ziehl-Neelsen stain for, 213 
Balantidium coli in feces, 179 

in gastric contents, 151 
Basophilic granulation of red blood 

cells, 303 
Beall's staining method, 221 
Bence- Jones' body in urine, 36 
Benedict's method of determination 
of glucose, first, 44 
second, 46 
Bial's test for pentose, 58 
Bile pigments in urine, 73. See 

also Bilirubin 
Bilharzia haematobium ( Schisto- 
soma haematobium), 118, 
192 
Bilirubin in feces, 158 
in urine, 73-76 

crystalline form of, 73, 94 
foam test for, 74 
Gmelin's test for, 74 



Bilirubin in urine, Hammarsten's 
test for, 75 
Huppert's test for, 75 
Rosenbach's test for, 74 
Bismarck brown, 220 
Bismuth in feces, 171 
Bjorn- Andersen and Lauritzen's 
formalin titration method, 
18 
Black's test for oxybutyric acid, 67 
Blastomycetes in sputum, 223 
Blood, the : 

anisocytosis, 302 

basophilic granulation of red 

cells, 303 
Boggs' coagulometer, 261 
Biirker's hemocytometer, 239 
Cabot's ring bodies, 304 
cleaning counting chamber, 234 
cleaning cover glasses, 265 
cleaning pipettes, 235 
cleaning slides, 265 
coagulation time, 260 
color index, 253 
counting, differential, 296 
of eosinophiles, 241 
of erythrocytes, 229 
of leukocytes, 236 
of platelets, 242 
differential counting, 296 
diluting fluids: 

for counting eosinophiles, 241 
for counting erythrocytes, 229 
for counting leukocytes, 236 
for counting platelets, 242 
Ehrlich's triacid stain, 280 
erythroblast, 302. See also Red 

cells, 
erythrocyte, 301. See also Red 

cells. 
Filaria bancrofti, 308 
films, preparation of, 268 



INDEX 



325 



Blood, the: 

fixation of blood films, 271 
fragmentation of nucleus, 303 
fresh blood, examination of, 266 
glassware, cleaning of, 265 
hematokrit, 253 
hemocytometer, Thoma's, 227 

Burker's modification of, 239 
hemoglobin estimation, 244 
Hess' viscosimeter, 256 
Howell's bodies, 303 
in feces, 153, 159, 169 
in gastric contents, 150 
in sputum, 210 
in urine, 80, 105 
intermediates, 302 
iodin reaction of leukocytes, 295 
irritation forms of Turk, 301 
Jenner's stain, 291 
karyorrhexis, 303 
labeling blood films, 271 
leukocytes, 297 

classification of normal and 
pathological, 297-301 
macrocyte, 301 
malarial parasites, 305 

in fresh blood, 308, 309 

staining for, 305 

Ross' method of, 306 
Ruge's modification of Ross' 
method of, 307 
mast cells, 299 
mast myelocyte, 300 
measurement of cells, 255 
megakaryocyte, 301 
megaloblast, 302 
megalocyte, 301 
metamyelocyte, 300 
methemoglobinemia, 252 
microblast, 302 
microcyte, 301 
micrometer, ocular, 255 



Blood, the : 

Miescher-Fleischl hemoglobinom- 

eter, 249 
Milian's method of determining 

coagulation time, 263 
mononuclear leukocytes, 298 
myeloblast, 300 
myelocyte, eosinophilic, 299 

"mast," 300 

neutrophilic, 299 
normoblast, 302 
normocyte, 301 
nuclear particles, 303 
obtaining blood for examination, 

226 
Pappenheim's methyl green-py- 

ronin stain, 295 
parasites, animal, 308 

Staubli's method of demon- 
strating, 310 
poikilocyte, 302 
polychromasia, 302 
polychromatophilia, 302 
polynuclear eosinophiles, 298 
polynuclear neutrophils, 298 
promyelocyte, 300 
protozoa in, 305 
red basophilic granulation, 304 
red cells, 301-305. 

abnormalities in staining, 302 

classification of normal and 
pathological, 301 

classification of nucleated, 302 
resistance of red cells, 264 
Rieder cells, 301 
ring bodies, 304 
Romanowsky stains, 283 
Sahli's hemometer, 245 
Schiiffner's granules, 304, 305 
smears, preparation of, 268 
specific gravity of blood, 259 

of serum, 260 



326 



INDEX 



Blood, the: 

staining the blood: 
dry films, 276 

carbol-thionin. 279 
Ehrlich's triaeid. 280 
eosin. 277 
hematoxylin. 277 
Jenner's stain. 291 
methyl green-pyronin. 2 
methylene blue. 276 
Eomanowsky stains. 2£ 
Gienisa's. 290 
Leishman's. 289 
Wilson's. 284 
triaeid stain of Ehrlieh, 28 
"vital" staining. 273 : 
dry method. 275 
Yaughan's method. 273 
Widal's method. 275 
Staubli's method of detecting 

parasites, 310 
stickers. 226 

stippling of red cells. 303 
sulph-hemoglobinemia. 252 
Tallqvist's color scale. 244 
Thoma's hemocytometer. 227 
transitional leukocytes. 2 9 
Trichinella spiralis. 310 
Tiirk's irritation forms, 301 
viscosity of blood. 256 
volume index, 253 
Boardinan's method for antiforaiin. 

217 
Boggs 1 coagulometer. 261 
Boggs' method of clearing para- 
sites. 201 
Borchardt's modification of Seli- 

wanoiFs test. 53 
Bothriocephalus latus. 196 
Brem's method of staining. 175 
Brodie and Russell's coagulometer. 
Boggs' modification of. 261 



Bromin solution. Rice's. B 

Bronchial casts, 206 

Burri's India ink method for 

spirochetes. 115 
Butyric acid in gastric contents. 142 



Cabot's ring bodies. 304 
Calcium carbonate in urine. 
Calcium oxalate in feces. 170 

in gastric contents. 151 

in urine. 92 
Calcium phosphate in urine. 97. 99 
Calcium soaps in feces. 170 
Calcium sulphate in urine. 93 
Calomel. 153 
Camphor as preservative of urine. 3 

- lie stain. Welch's. 219 
Carbol-fuehsin. 213 
Carbol-thionin. _" 
Casts, bronchial. 206 

urinary, 105 
Cercomonas hominis in feces. 177 

in gastric contents. 151 

in sputum. 224 
Cerebrospinal fluid, 31S-321 

bacteriology of. 319 

cells in. 31S 

counting cells in. 31 S 

globulin content of. 320 
Nbgachi test for. _ 
Ross-Jones test for, 321 

tubercle bacillus in. 319 

TVassermann reaction in, 321 
Cestodes (tapeworms). 194 
Charcot-Leyden crystals in feces, 
171 

in sputum. 211 
Cheese-mite in feces. 203 
Chlorids in mine. 23 
Chloroform as preservative of 
mine. 2 



INDEX 



327 



Cholesterin in chyluria, 85 

in echinococcus cysts, 225 

in feces, 170 

in urine, 94 
Chyluria, 84 

Cippolina's phenylhyclrazin test, 42 
Clap threads in urine, 109 
Clay-colored stools, 154 
Cleaning of counting chamber, 234 

of cover glasses, 265 

of hemometer, 249 

of pipettes, 235 

of slides, 265 
Clearing parasites, 200 
Clearing the urine, Kieselguhr in, 28 

lead acetate in, 48 
Coagulation time of the blood, 260 
Cochineal tincture, 23 
Collection of urine, 1 
Color index of red cells, 253 
Color of feces, 153 

of gastric contents, 129 

of sputum, 205 

of urine, 4 
Congo red test for hydrochloric 

acid, 134 
Connective tissue in feces, 167 
Corpora amylacea in urine, 119 
Counting, differential, 296 

of eosinophiles, 241 

of erythrocytes, 229 

of leukocytes, 236 

of platelets, 242 
Crystals, ammonio - magnesium 
phosphate, in feces, 170 
in urine, 98 

ammonium biurate, 98 

amorphous phosphates, 97 

amorphous urates, 91 

bilirubin (hematoidin), in feces, 
171 
in sputum, 212 



Crystals, bilirubin (hematoidin), 

in urine, 94 
bismuth suboxid, in feces, 171 
calcium carbonate, 97 
calcium oxalate, in feces, 170 

in urine, 92 
calcium phosphate, in feces, 170 

mono-, 93 

neutral, 99 

tri-, 97 
calcium sulphate, 93 
Charcot-Leyden, in feces, 171 

in sputum, 211 
cholesterin in chyluria, 85 

in echinococcus cyst, 225 

in feces, 170 

in urine, 94 
cystin, 96 
fatty acid, in feces, 171 

in sputum, 212 
hematoidin. See Bilirubin 
hippuric acid, 93 
leucin, 96 

magnesium phosphate, ammonio-, 
98 

neutral, 99 

tri-, 97 
microchemical reactions of, 99 
monocalcium phosphate, 93 
quadriurates of sodium and po- 
tassium, 91 
spermin, 119 
tricalcium phosphate, 97 
trimagnesium phosphate, 97 
triple phosphate, 98 
tyrosin, 94 
uric acid, 92 
xanthin, 94 
Curds in feces, 167 
Curschmann's spirals, 208 
Cylinders. See Casts, urinary 
Cylindroids, 109 



328 



INDEX 



D 

Deniges' test for tyrosin, 95 
Desmoid test of Sahli, 134 
Dextrose. See Glucose 
Diacetic acid in urine, 65 

Arnold's test for, 66 

Gerhardt's test for, 65 
Diastase. See Amylase 
Diazo reaction, 83 
Dibothriocephalus latus, 196 
Dicalcium phosphate in urine, 99 
Diluting fluid for eosinophils, 241 

for erythrocytes, 229 

for leukocytes, 236 

for platelets, 242 
Dimagnesium phosphate in urine, 

99 
Dimethylamidoazobenzole, 134 
Dioctophyme renale, 117 
Diphtheria bacilli, 219 
Diplococcus pneumonias, 217 
Dipterous larvae, 180 
Dipylidium caninum, 199 
Dittrich's plugs, 205 
Dock breakfast, 125 
Dunger's method of counting eosin- 
ophils, 241 
Dust cells in sputum, 210 



E 

Echinococcus, 225 
Ehrlich's aldehyde test, 70 
Ehrlich's diazo test, 83 
Ehrlich's hematoxylin, acid, 277 
Ehrlich's triacid stain, 280 
Elastic tissue in sputum, 208 
Entameba coli in feces, 176 
Entameba histolytica in feces, 172 

in sputum, 224 
Entameba tetragena in feces, 175 

in sputum, 224 



Enzymes. See Ferments 

Eosin, 277 

Eosinate of methylene blue (Jen- 

ner's stain), 291 
Eosinophilic cells, counting, 241 

in cerebrospinal fluid, 318 

in feces, 170 

in gastric contents, 150 

in puncture fluids, 316 

in sputum, 211 
Epithelial cells in feces, 169 

in gastric contents, 149 

in sputum, 209 

in urine, 100, 101 
Erythroblasts, 302 
Erythrocytes. See also Blood 

basophilic substances in, 302-304 

classification of pathological, 301 

color index of, 253 

counting, 229 

measuring, 255 

nucleated, 302 

resistance of, 264 

vital staining of, 273 

volume index of, 253 
Esbach's method of estimating al- 
bumin, 35 
Eustrongylus gigas. See Diocto- 
phyme renale 
Ewald breakfast, 125 



Fasciola hepatica, 192 

Fasciolopsis buskii, 193 

Fasting stomach, examination of, 

127 
Fat in feces, 154, 171 

in gastric contents, 150 

in sputum, 209 

in urine, 85, 86, 100, 107 
Fatty acid crystals in feces, 171 



INDEX 



329 



Fatty acid crystals in sputum, 212 
Feces : 

accidental contaminations, 203 
acholic stools, 154 
ammonio-magnesium phosphate 

crystals, 170 
amount, 153, 157 
Ankylostoma duodenale, 184 
Ascaris lumbricoides, 190 
Ascaris mystax (Toxocara canis), 

191 
Ascaris vermicularis ( Oxyuris 

vermicularis), 188 
Balantidium coli, 179 
bilirubin, 153, 157 
Gmelin's test for, 159 
Schmidt's test fV 158 
bismuth, 153, 171 
blood, 153, 159, 169 
guiac test for, 159 
hemin crystal test for, 161 
microscopic, 169 
Weber's test, 159 
calcium oxalate, 170 
calcium soaps, 170 
calomel, 153 

Cercomonas hominis, 177 
Charcot-Leyden crystals, 171 
cholesterin, 170 
clay-colored stools, 154 
clearing parasites, 199 
Boggs' method of, 201 
creosote method of, 203 
Mink and Ebeling's method of, 
200 
color, 153 

connective tissue, 167 
crystals, 170 
curds, 167 
debris, 168 

Dibothriocephalus latus, 196 
dipterous larvae, 180 
23 



Feces : 

Dipylidium caninum, 199 
Entameba coli, 176 
Entameba histolytica, 172 
Entameba tetragena, 175 
enzymes. See Ferments 
eosinophile cells, 170 
Fasciola hepatica, 192 
Fasciolopsis buskii, 193 
fat, 154, 171 
fatty acid, 171 
f rments, 161 

diastase (amylase), 163 

trypsin, 162 
fibrous connective tissue, 167 
food remnants, 167 
form, 153 
gall-stones, 155 
hookworm, 180-i86 
hydrobilirubin (urobilin), 153, 158 
Hymenolepis diminuta, 198 
Hymenolepis nana, 197 
iron, 153 

Lamblia intestinalis, 178 
leukohydrobilirubin, 154 
methylene blue, 153 
mucus, 154 
muscle fibers, 167 
Necator americanus, 180 

adult parasites of, 184 

embryo of, 181, 187 

ova of, 181 

special methods of finding ova 
of, 182-184 

special methods of finding 
parasites of, 184 
Opisthorchis sinensis, 192 
Oppler-Boas bacilli, 169 
Oxyuris vermicularis, 188 
Paragonimus westermanii, 194 
Paramecium coli (Balantidium 
coli), 179 



330 



INDEX 



Feces : 

parasites, intestinal, 172-203 

preservation of feces, 199 

preservation of parasites and ova 
in feces, 199, 200 

pus, 154, 170 

reaction, 157 

sareines, 169 

Schistosoma haematobium, 192 

Schistosoma japonicum, 193 

scybali, 153 

starch granules, 168 

Strongyloides stercoralis, 186 
ova of, 187 

rhabditiform embryo of, 186 
differentiation of, from 
hookworm embryo, 187 

tapeworms (cestodes), 194 

Tenia saginata, 194 

Tenia solium, 196 

test diets, intestinal, 155-157 

Toxocara canis, 191 

Trichinella spiralis, 191 

Trichocephalus dispar (Trichuris 
trichiura), 189 

Trichomonas intestinalis, 177 

Trichuris trichiura, 189 

triple phosphate, 170 

tubercle bacilli, 168 

Tyroglyphus siro, 203 

Uncinaria americana (Necator 
americanus), 180 

urobilin, 153, 158 

Schlesinger's test for, 158 
Schmidt's test for, 158 
spectroscopic determination of, 
158 

vegetable cells, 167 

vegetable hairs, 168 

vegetable spirals, 168 

weight of dried feces, 157 

yeasts, 169 



Fehling's test for sugar, 39 
Fermentation test for sugar, quali- 
tative, 41 
quantitative, 50-53 
Ferments in feces, 162-166 
in gastric contents, 143-148 
in urine, 86-89 
Fibrous connective tissue in feces, 

167 
Filaria bancrofti in blood, 308 

in urine, 117 
Films, blood, preparation of, 268 
Fischer's test meal, 126 
Fixation of blood films, 271 
Fleischl-Miescher hemoglobino- 

meter, 249 
Folin's method for acidity of urine, 
5 
for ammonia, 13 
Folin-Shaffer's method for uric 

acid, 10 
Food remnants in feces, 167, 152 

in gastric contents, 129, 149 
Formalin preservation of feces, 
199 
of urine, 3 
Formalin titration of ammonia, 16 
Functional diagnosis of the kidneys, 
119 
of pancreas, 162-166 
Futcher-Lazear fixation, 273 



G 

Gall-stones in stools, 155 
Garrod's test for hematoporphyrin, 

76 
Gastric contents: 
acetic acid, 143 
acidity : 

free acid, 136 
total acid, 138 



INDEX 



331 



Gastric contents: 

Balantidium coli, 151 
blood, chemical tests for, 159 

microscopic, 150 
butyric acid, 142 
Cercomonas hominis, 151 
color, 129 
crystals, 151 
eosinophile cells, 150 
fasting stomach, examination of, 

127 
fat droplets, 150 
ferments : 
pepsin, 143 

peptid-splitting ferment, 146 
rennin, 145 
food, 129 

glycyltryptophan test, 147 
hydrochloric acid, 131-138, 139 
deficit of, 139 
qualitative tests for: 
Congo paper test, 134 
Giinzberg's test, 132 
methyl violet test, 131 
Sahli's desmoid test, 134 
Toepfer's test, 134 
tropeolin test, 133 
quantitative determination of, 
136 
lactic acid, 140 

Kelling's test for, 142 
Strauss' test for, 141 
Ueffelmann's test for, 141 
Lamblia intestinalis, 151 
mucus, 128, 148 
odor, 128 

Oppler-Boas bacilli, 151 
pus, 150 

quantity of, after test meals, 128 
reaction, 131 
sarcines, 151 
starch, 149 



Gastric contents : 

test breakfasts and meals, 125- 
127 
Boas', 125 
Dock's, 125 
Ewald's, 125 
Fischer's, 126 
Riegel's, 126 
total acidity, 138 
Trichomonas intestinalis, 151 
yeasts, 150 
Gentian violet, anilin-water, 218 
Gerhardt's test for diacetic acid, 65 
Giemsa's stain for blood, 290 

for Treponema pallidum, 113 
Glassware, cleaning of, 265 
Globulin in cerebrospinal fluid, 320 
Noguchi's test for, 320 
Ross-Jones' test for, 321 
Glucose in urine, 38-53 
in chyluria, 85 
qualitative tests for, 38-44 
quantitative determination of, 44- 
53 
Glycerin jelly, 203 
Glycuronie acid in urine, 59 
Glycyltryptophan test, 147 
Gmelin's test for bile, 74 
Gonococcus in pus, 110 
Gram's iodin solution, 218 
Gram's stain, 218 

Gross' method of determining tryp- 
sin, 162 
Guiac test for blood in feces, 159 
in gastric contents, 159 
in urine, 81 
Gunning's test for acetone, 62 
Giinzberg's reagent, 132 

H 

Hammarsten's test for bilirubin, 75 
Hart's test for oxybutyric acid, 68 



332 



INDEX 



Harvey's modification of Volhard's 

method, 24 
Haser's coefficient, 7 
Hawk's modification of Wohlge- 

muth's method, 163 
Hayem's solution, 230 
Heart failure cells in sputum, 210 

in urine, 101 
Heller's test for albumin, 32 

for blood, 82 
Hematin, spectroscopic determina- 
tion of, 78 
Hematoidin (bilirubin) crystals in 
sputum, 212 
in urine, 94 
Hematokrit, 253 
Hematoporphyrin in urine, 76 
Hematoxylin, Ehrlich's acid, 277 
Hematuria, 80, 105 
Hemin crystal test of Teichmann 

for blood, 82 
Hemochromogen, 79 
Hemocytometer, Thoma's, 227 

Biirker's modification of, 239 
Hemoglobin, estimation of, 244-252 
in urine, tests for, 77, 81 
spectroscopic determination of, 
78 
Hemoglobin casts, 108 
Hess' viscosimeter, 256 
Hewlett's method of determining 

lipase, 88 
Hinman and Sladen's modification 

of Milian's method, 263 
Hippuric acid in urine, 93 
Hofmann's test for tryosin, 96 
Hookworm, 180-186: 

Ankylostoma duodenale, 184 
Necator americanus, 180 
Howell's bodies, 303 
Hufner's method of estimating 
urea, 8 



Huppert's test for bilirubin, 75 
Hydrobilirubin (urobilin) in feces, 

153, 158 
Hydrochloric acid in gastric con- 
tents, 131 
deficit of, 139 

qualitative tests for, 131-135 
quantitative determination of, 136 
Hymenolepis diminuta, 198 
Hymenolepis nana, 197 
Hypobromite method of estimating 
urea, 8 



Incoagulable nitrogen in puncture 

fluids, 313 
India ink method for spirochetes, 

115 
Indican in urine, Jaffe's test for, 
27 
Obermayer's test, 27 
Indicators : 

alizarin red, 13, 15 
cochineal tincture, 23 
Congo red, 134 
dimethylamidoazobenzole (Toep- 

fer's reagent), 134 
Giinzberg's reagent, 132 
phenolphthalein, 138 
Indoxyl sulphate {See Indican), 27 
Influenza bacilli, 219 
Intermediate nucleated red cells, 

302 
Iodin reaction of leukocytes, 295 
Iron in stools, 153 
Irritation forms of Turk, 301 



Jaffe's test for indican, 27 

for urobilin, 72 
Jenner's stain, 291 



INDEX 



333 



K 

Karyorrhexis, 303 

Kieselguhr, 28 

Kjeldahl's method of determining 
total nitrogen, 20 



Labeling blood films, 271 

Lactic acid in gastric contents, 140 

Lactose in urine, 56 

Lamblia intestinalis in feces, 178 

in gastric contents, 151 
Lange's test for acetone, 64 
Large mononuclear leukocytes, 298 
azurophilic granules in, 287, 298 
characteristics of, 298 
staining reactions of, with Jen- 
ner's stain, 293, 294 
with triacid stain, 283, 294 
with Wilson's stain, 287, 294 
Lecithin in chyluria, 85 
in prostatic fluid, 119 
Legal's test for acetone, 64 
Leishman's stain, 289 
Leucin in urine, 96 
Leukocytes. See also Blood 
classification of normal, 297 
classification of pathological, 299 
counting, 236 
counting eosinophilic, 241 
differential count of, 296 
iodin reaction of, 295 
measuring, 255 

staining reactions of, with Jen- 
ner's stain, 293, 294 
with triacid stain, 282, 294 
with Wilson's stain, 287, 294 
vital staining of, 273 
Leukohydrobilirubin in feces, 154 
Levulose in urine, 53 

phenylhydrazin test for, 55 



Levulose in urine, quantitative de- 
termination of, 46 
Seliwanoff's test for, 53 
Levulosuria, alimentary, 56 
Lieben's test for acetone, 63 
Lipase in urine, 88 
Lipuria, 86 

Loeffler's antiformin method, 215 
Loeffler's methylene blue, 213 
Lohnstein's areometer, 51 
Lohnstein's fermentation saccha- 

rometer, 52 
Lugol's solution, 63 
Lymphocytes, 297 

azurophilic granules in, 287, 297 
characteristics of, 297 
in cerebrospinal fluid, 318 
in chyluria, 86 
in puncture fluids, 316 
in sputum, 211 
pathological, 301 
staining reactions of, with Jen- 
ner's stain, 293, 294 
with methyl green-pyronin 

mixture, 295 
with triacid stain, 283, 294 
with Wilson's stain, 287, 294 



M 

Macrocyte, 301 

Macrophage ( large mononuclear 

leukocyte), 298 
Magnesium phosphate in urine, 97, 

98, 99 
Malarial parasites, 305 
differential table of, 309 
in fresh blood, 308 
Schiiffner's granules, 305 
stained by carbol-thionin, 279 
by Romanowsky stains, 283, 
285, 290 



334 



INDEX 



Malarial parasites, stained by car- 
bol-thionin, by Ross' 
method, 306 
by Ruge's modification of the 
method of Ross, 307 
Maltose in urine, 56 
Mast cells: 

characteristics of, 299 
staining reactions of, with carbol- 
thionin, 279 
with Jenner's stain, 293, 294 
with methylene blue, 276 
with triacid stain, 282, 294 
with Wilson's stain, 287, 294 
Mast myelocyte, 300 
Measurement of microscopic ob- 
jects, 255 
Megakaryocyte, 301 
Megaloblast, 302 
Megalocyte, 301 
Metamyelocyte, 300 
Methemoglobin in blood, 252 

in urine, 78 
Methyl green-pyronin stain, 295 
Methyl violet test for hydrochloric 

acid, 131 
Methylene blue in feces, 153 

in urine (following the desmoid 

test), 135 
Loeffier's, 213 
Mette's method for determination 

of pepsin, 144 
Microblast, 302 
Microcyte, 301 
Micrometer, ocular, 255 
Microphage (polynuclear neutro- 
philic leukocyte), 298 
Miescher-Fleischl hemoglobinom- 

eter, 249 
Milian's method of determining co- 
agulation time, 263 
Millon's reagent, 96 



Mink and Ebeling's method of 

clearing parasites, 200 
Monocalcium phosphate in urine, 

93 
Mucous threads in urine, 109 
Mucus in feces, 154 

in gastric contents, 128, 148 
Murexid test, 9 
Muscle fibers in feces, 167 
Myelin degeneration of epithelial 
cells in sputum, 210 

in urine, 101 
Myeloblast, 300 
Myelocyte, eosinophilic, 299 

mast (basophilic), 300 

neutrophilic, 299 

staining reactions, 294 

N 
Necator americanus, 180 

adult worms of, 184 

embryo of, 181, 187 

ova of, 181 

special methods of finding ova 
of, 182 
of finding parasites of, 184 
Neisser's stain, 220 
Nematodes, 180 
Nitrogen, incoagulable, 313 

total, 20 
Noguchi's globulin test, 320 
Normoblast, 302 
Normocyte, 301 
Nubecula in urine, 3 
Nuclear particles, 303 
Nylander's test for sugar, 40 

O 

Obermayer's test for indiean, 27 
Opisthorchis sinensis, 192 
Oppler-Boas bacilli in feces, 169 
in gastric contents, 151 



INDEX 



335 



Ova. See the various parasites 
special methods of detecting, 182 : 
centrifugalization, 183 
Pepper's method, 182 
Stiles' method, 182 
Oxybutyric acid (/?), Black's test 
for, 67 
Hart's test, 68 
Oxyhemoglobin in urine, 78 



Pappenheim's stain, 295 
Paragonimus westermanii in feces, 
194 

in sputum, 224 
Paramecium eoli (Balantidium 

coli), 179 
Parasites in blood, 308 

in feces, 172-199 

in sputum, 224 

in urine, 116 
Paterson's antiformin method, 216 
Pentose in urine, 57 

Bial's orcin test for, 58 

orcin test for, 58 

phloroglucin test for, 57 
Pepper's method of detecting hook- 
worm ova, 182 
Pepsin in gastric contents, 143 
Peptid-splitting enzyme in gastric 

contents, 146 
Pericardial fluids, 312 
Peritoneal fluids, 312 
Phenolphthalein, 138 
Phenolphthalin, 82 
Phenolsulphonephthalein test, 120 
Phthalein test, 120 
Pinworm ( Oxy uris vermicularis ) , 

188 
Piria's test for tyrosin, 95 
Platelets, blood, counting, 242 



Platelets, blood, staining reactions 
of, with Wilson's stain 
(Romanowsky), 287, 294 
Pleural fluids, 312 
Pneumococcus in sputum, 217 
Poikilocyte, 302 

Polariscopic determination of sug- 
ars, 48 
Polychromasia, 302 
Polychromatophilia, 302 
Polynuclear eosinophiles, character- 
istics of, 298 
staining reactions of, with eosin, 
277 
with Jenner's stain, 293, 294 
with triacid stain, 282, 294 
with Wilson's stain, 287, 294 
Polynuclear neutrophiles, charac- 
teristics of, 298 
staining reactions of, with Jen- 
ner's stain, 293, 294 
with triacid stain, 282, 294 
with Wilson's stain, 287, 294 
Preservation of animal parasites, 
199, 200 
of feces, 199 
of urine, 2 
Promyelocyte, 300 
Prostatic fluid, 119 

corpora amylacea in, 119 
lecithin in, 119 
spermatozoa in, 119 
spermin crystals in, 119 
Protein precipitable by acetic acid 

in cold, 315 
Pseudocast, 109 

Puncture fluids (pleural, peritoneal, 
pericardial), 312 
albumin content of, 312 

Kjeldahl determination of, 313 
Tsuchiya's reagent for, 312 



336 



INDEX 



Puncture fluids, incoagulable nitro- 
gen in, 313 
protein in, precepitable by acetic 

acid in cold, 315 
serosaniucin in, 315 
specific gravity of, 312 
tubercle bacilli in. 317 
Pus in feces. 151. 170 
in gastric contents, 150 
in sputum. 211 
in urine. 102 

counting cells of. 103 

false albuminuria due to, 101 

guiae tests for, 104 



Q 

Quadriurates of sodium and potas- 
sium, 91 
Quantity of feces, dried. 157 
in twenty-four hours. 153 
of gastric contents after test 
meals. 12S 
from fasting stomach, 127 
of urine in twenty-four hours, 4 



E 

Reaction of feces. 157 

of gastric contents, 131 

of sputum, 205 

of urine. 1 
Red basophilic granulation, 304 
Red blood cells. See Erythrocytes 

and Blood 
Reduced hematin. See Hemochro- 

mogen 
Reduced hemoglobin. 75 
Removal of albumin from urine, 36 
Resistance of erythrocytes, 264 
Rice's brormn solution, S 
Rieder cells. 301 



Riegel's dinner. 126 

Ring bodies, 304 

Robert's specific gravity fermenta- 
tion method. 50 

Romanowsky stains for blood, 2S5, 
2-9. 290 
for Treponema pallidum. 112 

Ronehese, formalin titration of am- 
monia. 17 

Rosenbach's test for bile. 74 

Ross' method of detecting malarial 
parasites, 306 

Roundworms (nematodes). 180 

Rowntree and Geraghty's phthalein 
test. 120 

Ruge's modification of Ross' 
method, 307 



Saccharose in urine. 57 
Sahli's desmoid test. 134 
Sahli's hemometer. 245 
Sarcines in feces, 169 

in gastric contents, 151 
Schistosoma haematobium in feces, 

192 
in urine, 118 
Schistosoma japonicum. 193 
Schlesinger's test for urobilin in 

feces. 158 
in urine, 71 
Sehlosing's method of determining 

ammonia. 19 
Schmidt and Strasburger's test 

diets, 156 
Schmidt's test for bilirubin, 158 

for urobilin. 158 
Schuftners granules, 305 
Schmnm's method of spectroscopic 

examination, 80 
Scvbali. 153 



INDEX 



337 



Seatworm (Oxyuris vermicularis), 

188 
Sediments in urine, organized, 100 

unorganized, 91 
Seliwanoff's test for levulose, 53 
Serosamucin, 315 
Shaffer's vacuum distillation of 

ammonia, 15 
Silver impregnation method for 

spirochetes, 114 
Simon's method of staining amebae, 

174 
Smears, blood, 268 
Smegma bacillus in urine, 115. See 

under Tubercle bacillus 
Specific gravity of blood, 259 
of blood plasma, 260 
of blood serum, 260 
of puncture fluids, 312 
of urine, 6 
Spectra for glycuronic acid com- 
pounds (Tollens' test), 60 
for hematin, acid, 78 
for hematoporphyrin, 76 
for hemochromogen, 79 
for hemoglobin, reduced, 78 
for methemoglobin, 78 
for oxyhemoglobin, 78 
for pentose (orcin test), 58 
for urobilin, 71 
Spermatozoa in urine, 119 
Spermin crystals, 119 
Sputum : 

Actinomyces bovis, 221 
alveolar epithelial cells, 209 
amount, 205 

antiformin method for detecting 
tubercle bacilli, 214-217 
Boardman's, 217 
Loeffler's, 215 
Paterson's, 216 
blastomycetes, 223 



Sputum : 

blood, 210 

bronchial casts, 206 

bubbles in, 205 

Cercomonas hominis, 224 

character, 205 

Charcot-Leyden crystals, 211 

consistence, 205 

Curschmann's spirals, 206, 208 

diphtheria bacilli, 219 

Beall's method of staining, 221 
Neisser's stain for, 220 

Diploeoccus pneumoniae, 217 

Dittrich's plugs, 205 

dust cells, 209, 210 

echinococcus, 225 

elastic tissue, yellow, 208 

Entameba histolytica, 224 

Entameba tetragena, 224 

eosinophile cells, 211 

fat droplets, 209 

fresh sputum, plate method of 
examination, 207 

heart failure cells, 210 

influenza bacilli, 219 

layer formation, 206 

lymphocytes, 211 

myelin degeneration of alveolar 
cells, 210 

obtaining sputum, 204 . 

odor, 205 

Paragonimus westermanii, 224 

plate method of examination, 207 

pneumococcus, 217 

Welch's capsule stain for, 219 

pus, 211 

reaction, 205 

streptothrix, 222 

Trichomonas intestinalis, 224 

tubercle bacilli, 212 

antiformin methods for, 214-217 
Ziehl-Neelsen stain for, 213 



338 



INDEX 



Stains : 

anilin-water gentian violet, 218 
Beall's method of staining diph- 
theria bacilli, 221 
Bismarck brown (vesuvin), 111 
Brem's method for staining 
amebae, 175 

for old blood films, 290 
capsule stain, Welch's, 219 
carbol-fuchsin, 213 
carbol-thionin, 279 
Ehrlich's hematoxylin, acid, 277 
Ehrlich's triacid stain, 2S0, 294 
eosin, 277 

gentian violet, anilin water, 218 
Giemsa's stain for blood, 290 

for old smears, 290 

for spirochetes, 113 
Gram's stain, 218 
hematoxylin, Ehrlich's acid, 277 
Jenners stain, 291, 291 
Leishman's stain, 289 
Loeffler's methylene blue, 213 
methyl green-pyronin, 295 
methylene blue, Loeffler's, 213 
neutral red, 174, 276 
Neisser's stain, 220 
Pappenheim's methyl green-py- 
ronin, 295 
polychrome methylene blue, 

Unna's, 273 
Romanowsky stains for blood, 
283, 285, 289, 290, 294 

for old smears, 290 

for spirochetes, 112 
Scharlach R, 171 
Simon's method of staining 

amebae, 174 
Sudan III, 171 
triacid stain of Ehrlich, 280 
vesuvin (Bismarck brown), 111 
Welch's capsule stain, 219 



Stains: 

Wilson's stain, 285 

Ziehl-Neelsen stain, 213 
Starch granules in feces, 168 

in gastric contents, 149 
Staubli's method of detecting para- 
sites, 310 
Stern's silver impregnation method, 

114 
Stiles' method of detecting hook- 
worm ova, 182 
Stippling of red cells, 303 
Stomach contents. See Gastric con- 
tents 
Stools. See Feces 
Strongyloides stercoralis, 186 

embryo of, rhabditiform, 186 

ova of, 187 
Sulphates in urine (indican), 26 
Sulph-hemoglobinemia, 252 



Tallqvist's color scale, 244 

Tapeworms (cestodes), 194 

Teichmann's hemin crystal test, 82 

Tenia saginata, 194 

Tenia solium, 196 

Test breakfasts and meals, 125 

Boas', 125 

Dock's, 125 

Ewald's, 125 

Fischer's, 126 

Riegel's, 126 

Schmidt and Strasburger's, 156 
Test diet, intestinal, 155: 

diet No. I, 156 

diet No. II, 156 
Thymol as preservative of urine, 3 
Toepfer's test for hydrochloric 

acid, 134 
Toisson's solution, 230 
Toluol (toluene) as preservative of 



urine, 2 



INDEX 



339 



Toxocara canis, 191 
Transitional leukocytes, 298 
azurophilic granules in, 298 
characteristics of, 298 
staining reactions of, with Jen- 
ner's stain, 293 
with triacid stain, 283 
with Wilson's stain, 287 
Trematodes, 192 
Triacid stain of Ehrlich, 280 
Tricalcium phosphate in urine, 97 
Trichinella spiralis in blood, 310 
Staubli's method of detecting, 
310 
in feces, 191 
Trichocephalus dispar ( Trichuris 

trichiura), 189 
Trichomonas intestinalis in feces, 
177 
in gastric contents, 151 
in sputum, 224 
Trichomonas vaginalis in urine, 116 
Trichuris trichiura, 189 
Trimagnesium phosphate in urine, 

97 
Triple phosphate in feces, 170 
in gastric contents, 151 
in sputum, 212 
in urine, 98 
Tripperfaden in urine, 109 
Trommer's test for sugar, 38 
Tropeolin test for hydrochloric acid, 

133 
Trypsin in feces, 162 
Tsuchiya's method for albumin in 
puncture fluids, 312 
in urine, 35 
Tubercle bacillus in cerebrospinal 
fluid, 319 
in feces, 168 
in puncture fluids, 317 
in sputum, 212 



Tubercle bacillus in urine, 115 

Ziehl-Neelsen stain for, 213 
Tumor cells in puncture fluids, 316 
Turk's irritation forms, 301 
Tyroglyphus siro, 203 
Tyrosin in urine, 94 

Deniges' test for, 95 

Hofmann's test for, 96 

Piria's test for, 95 



U 



Uncinaria americana (Necator 

americanus), 180 
Urea, 7 

Uric acid in urine, 9 
crystals of, 92 
microchemical reactions of, 92, 

99 
murexid test for, 9 
quantitative determination of, 10. 
Urine : 

acetone, 62 

Gunning's test for, 62 
Lange's test for, 64 
Legal's test for, 64 
Lieben's test for, 63 
acidity, 4 

Folin's method of determina- 
tion of, 5 
albumin, 28 

heat and acetic acid test for, 28 
heat and nitric acid test for, 31 
Heller's test for, 32 
potassium ferrocyanid and 

acetic acid test for, 34 
quantitative determination of, 

35 
removal of, 36 
albumose, 37 
alkaptonuria, 61 
ammonia, 12 



340 



INDEX 



Urine: 

ammonia, Folin's method of de- 
termination of, 13 
formalin titration methods of 

determination of, 16 
Schlosing's method of deter- 
mination of, 19 
Shaffer's method of determina- 
tion of, 15 
ammonio-magnesium phosphate, 

98 
ammonium biurate, 98 
bacteria : 

gonococcus, 110 
smegma bacillus, 115. See 
under Tubercle bacillus 
tubercle bacillus, 115 
Bence-Jones' body, 36 
bile pigments (bilirubin), 73 
bilirubin (hematoidin), 73 
crystals of, 73, 94 
foam test for, 74 
Gmelin's test for, 74 
Hammarsten's test for, 75 
Huppert's test for, 75 
Rosenbach's test for, 74 
staining of sediment by, 74, 101 
blood (hematuria), 80 
cells of, in sediment, 105 
guiac test for, 81 
Heller's test for, 82 
hemin crystal test for, 82 
calcium carbonate, 97 
calcium oxalate, 92 
calcium phosphate, 93, 97, 99 
calcium sulphate, 93 
casts, 105 
amyloid, 108 
blood, 106 

coarsely granular, 107 
colloid, 108 
epithelial, 106 



Urine : 

casts, fatty, 107 

finely granular, 107 
hemoglobin, 108 
hyalin, 108 
pus, 106 
waxy, 108 
chlorids, 23 
cholesterin, 94, 85 
chyluria, 84 
clap threads, 109 
clearing, Kieselguhr in, 28 

lead acetate in, 48 
collection of urine, 1 
color, 4 

corpora amylacea, 119 
cylinders. See Casts 
cylindroids, 109 
cystin, 96 

dextrose {See Glucose), 38 
diacetic acid, 65 
diazo reaction, 83 
Dioctophyme renale, 117 
Ehrlich's aldehyde test, 70 
Ehrlich's diazo test, 83 
epithelial cells, 100 
Eustrangylus gigas (Diocto- 
phyme renale), 117 
Filaria bancrofti, 117 
functional diagnosis, phthalein 

test, 119, 120 
glucose, 38 

qualitative tests for: 

Almen-Nylander's test, 40 
Fehling's test, 39 
fermentation test, 41 
phenylhydrazin test, 42 
Trommer's test, 38 
quantitative determination of: 
Benedict's first method, 44 
Benedict's second method, 46 
fermentation, gas, 51 



INDEX 



341 



Urine : 

glucose, quantitative determina- 
tion of: 
fermentation, specific grav- 
ity, 50 
polariscopic, 48 
glycuronic acid, 59 

Tollens' test for, 60 
gonococcus, 110 

grape sugar (See Glucose), 38 
Haser's coefficient, 7 
heart failure cells, 101 
hematin, acid, 78 
reduced (see hemochromogen), 
79 
hematoporphyrin, 76 
hemochromogen, 79 
hemoglobin, 77 
guiac test for, 81 
Heller's test for, 82 
spectroscopic determination of, 

78 
Teichmann's hemin crystal test 
for, 82 
hippuric acid, 93 
indie an, 26 

Jaffe's test for, 27 
Obermayer's test for, 27 
indoxyl sulphate (See Indican), 

26 
lactose, 56 
lecithin in chyluria, 85 

in prostatic fluid, 119 
leucin, 96 
levulose, 53 

phenylhydrazin test for, 55 
quantitative determination of, 

46 
Seliwanoff's test for, 53 
magnesium phosphate, neutral, 99 
maltose, 56 
methemoglobin, 78 



Urine: 

microchemical reactions of crys- 
tals, 99 
microscopic examination, 89 

specimens for, 1, 89 
monocalcium phosphate, 93 
mucous threads, 109 
nitrogen, total 20 
nubecula, 3 

obtaining sediments, 89 
oxybutyric acid, /?-, 67 

Black's test for, 67 

Hart's test for, 68 
oxyhemoglobin, 78 
parasites, animal, 116: 

Dioctophyme renale, 117 

Filaria bancrofti, 117 

Schistosoma haematobium, 118 

Trichomonas vaginalis, 116 
pentose, 57 

orcin test for, 58 
modified by Bial, 58 

phloroglucin test for, 57 
phenolsulphonephthalein test, 120. 

See also Phthalein test 
phthalein test, 120 
preservation of urine, 2 
prostatic fluid, 119 
pseudocasts. 109 
pus, 102 

counting cells of, 103 

false albuminuria from, 104 

guiac test for, 104 

Meyer's modification, 104 

microscopic, 102 
quadriurates of sodium and po- 
tassium, 91 
quantity for twenty-four hours, 4 
reaction, 4 
saccharose, 57 

Schistosoma haematobium, 118 
sediments, 89 



342 



INDEX 



Urine: 
sediments : 

ammonio-magnesium phos- 
phate, 98 

ammonium biurate, 98 

bilirubin, 94 

blood cells, 105 

calcium carbonate, 97 

calcium oxalate, 92 

calcium phosphate, mono-, 93 
neutral, 99 

calcium sulphate, 93 

casts, 105 

cholesterin, 94 

clap threads, 109 

cylindroids, 109 

cystin, 96 

epithelial cells, 100 

heart failure cells, 101 

hematoidin, 94 

hippuric acid, 93 

leucin, 96 

magnesium phosphate, neutral, 
99 

monocalcium phosphate, 93 

mucous threads, 109 

obtaining sediments, 89 

pseudocasts, 109 

pus, 102 

quadriurates of sodium and po- 
tassium, 91 

tricalcium phosphate, 97 

trimagnesium phosphate, 97 

triple phosphate, 98 

Tripperfaden, 109 

tyrosin, 94 

uric acid, 92 

xanthin, 94 
smegma bacillus, 115. See under 

Tubercle bacillus 
specific gravity, 6 
spermatozoa, 119 



Urine : 

spermin crystals, 119 

Spirochseta pallida, 111 

Spirochaeta refringens, 112 

sulphates. See Indican 

Treponema pallidum, 111 

tricalcium phosphate, 97 

trimagnesium phosphate, 97 

triple phosphate, 98 

Tripperfaden, 109 

tubercle bacillus, 115 

tyrosin, 94 

urea, 7 

uric acid, 9 
crystals of, 92 
murexid test for, 9 
quantitative determination of, 
10 

urobilin, 70 

Jaffe's test for, 72 
Schlesinger's test for, 71 
spectroscopic determination of, 
71 

urobilinogen, 70 

xanthin, 94 
Urobilin in feces, 153, 158 

in urine, 70 
Urobilinogen in urine, 70 



Vacuum distillation of ammonia, 15 
Vegetable cells in feces, 167 
Vegetable hairs in feces, 168 
Vegetable spirals in feces, 168 
Vesuvin (Bismarck brown), 111 
Viscosimeter of Hess, 256 
Viscosity of blood, 256 
Volhard's method for determination 

of chlorids, 24 
Volume irdex of red cells, 253 
von den Velden's test for hydro- 
chloric acid, 131 



INDEX 



343 



W 

Wassermann reaction in cerebro- 
spinal fluid, 321 

Weber's test for blood, 159 

Weidel's test for xanthin, 94 

Welch's capsule stain, 219 

Whipworm (Trichuris trichiura), 
189 

Wilson's stain, 284 

Wohlgemuth's method for diastase 
in feces, 163 
in urine, 87 

Wright and Kinnicutt's method of 
counting platelets, 242 



Xanthin in urine, 94 



Yeasts in feces, 169 

in gastric contents, 150 
Yellow elastic tissue, 208 



Zenker's fluid, 200 
Ziehl-Neelsen stain for 
bacilli, 213 



tubercle 



(i) 



APR 3.3 1913 



